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Detection of Histatin 5 in Normal Human Schirmer Strip Samples by Mass Spectroscopy

^A Ophthalmology^B Pharmacology and Toxicology¹ University of Louisville Louisville KY

Commercial Relationships:  P.S. Steele, None; M.M. Jumblatt, None; N.B. Smith, None; W.M. Pierce, None.
Grant Identification: NIH 1R03 EY13860-01, NIH/NEI EY 10736, NIH/NIDR T-32 DE07254, The Kentucky Lions Foundation, an unre

Abstract

Purpose: Antimicrobial proteins, including MUC7, defensins, and histatins, are products of the oral mucosa and participate in the non-immune defense system that protects the oral cavity from opportunistic infection. Using RT-PCR, we have shown that histatin, like MUC7 and defensins, is present in ocular tissue. Cationic gel electrophoresis coupled with both tryptic hydrolysis and mass spectrometry demonstrated that mass spectral results were consistent with the presence of histatin in fractionated "whole" tears. Tear sampling and low protein concentrations create specific challenges in the assessment of tear film. Utilization of tear samples directly from Schirmer strips collected as part of routine ocular surface exams would be particularly advantageous in the acquisition of tear samples without repeated or extraneous sampling methods, especially in patients with low tear volumes. We therefore explored the possibility of extracting tears directly from Schirmer strips for histatin analysis. Methods: Tears were collected from normal volunteers. Tears were eluted from dried Schirmer strips in a methanol: water mixture (75:25) with 0.1% formic acid. An equal volume of an acid pretreatment was added to remove higher molecular weight proteins. Following centrifugation, the supernatant was removed, evaporated, and reconstituted in de-ionized water. Samples were analyzed by RP-HPLC and electrospray mass spectrometry. Results: Mass spectral data positively identified the presence of histatin in tear samples eluted from Schirmer strips. Sample retention time was identical to that of the histatin standard. Conclusion: We have identified histatin as a component of tear film following elution from Schirmer strips. The ability of mass spectrometry to detect histatin in tear samples collected on Schirmer strips suggests that this method may be useful for determining the abundance of histatin and other tear film proteins in dry eye patients. Support: NIH 1R03 EY13860-01, NIH/NEI EY 10736, NIH/NIDR T-32 DE07254, The Kentucky Lions Foundation, an unrestricted grant from the Research to Prevent Blindness, NIH 1S10RR11368-01A1 (Biomolecular Mass Spectrometry Lab), State of Kentucky Physical Facilities Trust Fund, UofL School of Medicine, and the UofL Research Foundation.

Keywords: 376 cornea: tears/tear film/dry eye

© 2002, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}arvo.org.