Human lipocalin-1 association with ³H-testosterone and ³H-estradiol

Purpose: Topical androgens and estrogens have been studied for use in treating ocular conditions such as dry eye. The aim of this study was to identify proteins from normal human tears that associated with exogenously added sex steroid hormones. One of the major proteins in ocular tears is lipocalin-1. It binds a variety of lipids and other hydrophobic molecules and is proposed to function as a carrier protein or a lipid scavenger. Methods: Normal human tears were incubated with ³H-testosterone or ³H-estradiol. Labeled tear proteins were separated on a Q Sepharose Fast Flow (QFF) Hi Trap strong anion exchange column with a step gradient of NaCl. ³H- testosterone or ³H-estradiol was measured in aliquots of eluted fractions using scintillation counts, and the remainder of each sample was gel electrophoresed and silver stained. In separate experiments, ³H-steroid-labeled tear proteins were electrophoresed in 15% polyacrylamide gels and excised from the gels. Tritium content of the proteins was measured in a scintillation counter. Immunoblots with antibodies to lipocalin-1 verified the migration of lipocalin-1 in the gels. Results: ³H-steroid labeled tear proteins were found in the 0.15M NaCl fractions of QFF strong anion exchange columns. 18 kD lipocalin-1 (among other tear proteins) eluted in the 0.15M NaCl fraction. Excision of labeled tear proteins from 15% polyacrylamide gels indicated that radioactive label was associated with an 18 kD protein. Immunoblots verified that lipocalin-1 migrated as an 18 kD protein. Conclusions: The sex steroid hormones testosterone and estradiol associated with 18 kD lipocalin-1 in human tears.