Purpose: Dry eye is a collection of diseases characterized by insufficient ocular tears. Androgens play a vital role in tear production and ocular surface health. A current hypothesis is that topical androgen treatments may alleviate symptoms of dry eye, however, little is known about sex steroid hormones in the tears. We asked which tear protein(s) bind steroid hormones in isolated normal human tear samples.
Methods: Tears were collected from healthy volunteers. 100µl tears were incubated with 50 nM or 200 nM 3H-testosterone or 3H-estradiol at 37°C for 90 minutes. Free steroid was removed with dextran-coated charcoal.1) Ion exchange column (IEC) chromatography: In one set of experiments, 3H-Labeled tears were loaded on a 1 ml QFF HiTrap IEC in 20 mM TrisHCl, pH 6.0. Proteins were eluted in TrisHCl, pH 6.0 with a range of NaCl from 0.05M - 0.4M. 100µl of each 1 ml elution were counted in scintillation fluid. Proteins in the remaining 900µl were TCA precipitated, reduced, electrophoresed in 15% polyacrylamide gels and silver stained.2) Gel fragments: In a second set of experiments, 10µl 3H-labeled tears were loaded per lane and electrophoresed in 15% polyacrylamide gels. Gel fragments from silver stained or unstained gels were retrieved, dissolved in 30% hydrogen peroxide, and counted in scintillation fluid.
Results: Pooled tear samples bound 2-3% of 3H-testosterone, and 4-8% of 3H-estradiol.1) IEC chromatography: 3H-testosterone and 3H-estradiol fractions from the QFF IEC coincided with the elution profile of lipocalin-1. Other proteins were also present in these fractions.2) Gel fragments: 15-30% of the bound 3H-label was recovered after SDS gel electrophoresis. Gel fragments from reduced and non-reduced protein samples contained 3H-labeled protein at 18 kD.
Conclusions: Lipocalin-1 is one of the major proteins in ocular tears. Lipocalin-1 binds a variety of lipids and hydrophobic molecules, and is proposed to function as a carrier protein or a lipid scavenger. Lipocalin-1 is present in tears as a 37 kD dimer in equilibrium with an 18 kD monomer. Our data show that the monomer of lipocalin-1 nonspecifically binds 3H-testosterone and 3H-estradiol. Our data imply that lipocalin-1 acts as a scavenger for steroid hormones in tears.