SHORT COMMUNICATION Detection of substance P in human tears by laser desorption mass spectrometry and immunoassay

Abstract

PURPOSE. To determine whether substance P is present in human tears. METHODS. Tear samples (1–2 µl) were collected from one eye of each of 12 subjects. Two of the eyes had dry eye syndrome, two wore contact lenses and had dry eye syndrome, and eight were normal. Five of the eight normal eyes were scheduled to undergo excimer laser refractive surgery, and tears were collected from these eyes before and after surgery. Tear samples were analyzed by laser desorption mass spectrometry. Pooled samples from one individual were subjected to enzyme-linked immunoabsorbent assay. RESULTS. Laser desorption mass spectra of the 18 tear samples displayed well defined peaks with mass to charge (m/z) ratios ranging from 1343.7 to 1355.9 and/or 1356.9 to 1364.7, corresponding to an average m/z of 1349.8 ± 1.13 for protonated substance P and 1361.2 ± 0.54 for oxidized substance P obtained from 14 mass spectra of standards formulated with substance P concentrations ranging from 10 -4 M to 10 -12 M. As confirmation, an enzyme-linked immunoabsorbent assay performed twice on pooled tears from one eye detected substance P in both replicates at a concentration of 125 pg/ml (9.26 × 10 -11 M). CONCLUSIONS. These findings demonstrate that substance P is a component of tears obtained from normal eyes of men and women ranging in age from 26 to 60 years, from eyes fitted with contact lenses, from eyes with dry eye syndrome, and from eyes 1 and 2 days after excimer laser refractive surgery. Whether the concentration of substance P in tears varies with sex, age, or eye condition, the source of substance P in tears, and its role in tears remains to be discovered.