Changes in human tear protein levels with progressively increasing stimulus

The levels of 13 proteins were measured in six tear samples collected atraumatically at progressively increasing flow rate from nonstimulated (less than 0.5 microliter/min) to highly stimulated (greater than 50 microliters/min) in ten subjects. Tears were fractionated initially by size-exclusion high-performance liquid chromatography (SE-HPLC). Enzyme-linked immunosorbent assays and kinetic assays were then applied to relevant SE-HPLC fractions to determine specific protein levels. Nine of the 13 proteins assayed showed significantly higher concentrations in nonstimulated tears than in any other tear sample. Immunoglobulin (Ig) M, secretory IgA, polymeric IgA1, and polymeric IgA2 all decreased progressively in concentration from nonstimulated tears to the higher flow-rate stimulated samples. The level of IgG, albumin, and transferrin showed a large drop in concentration between nonstimulated tears and the first (lowest flow-rate) stimulated sample, with relatively little decrease for any subsequent sample. Levels of lactoferrin, tear-specific prealbumin, lysozyme, and peroxidase were relatively constant throughout the series of tear samples. These results indicate that the mechanisms responsible for changes in concentration of constitutive, serum-derived, and regulated tear proteins with stimulus can be studied successfully using noninvasive methods to collect human tears. They also show that simply distinguishing between nonstimulated and stimulated tears is not sufficient to completely characterize the effect of stimulus conditions on tear protein composition.