DETERMINATION OF PHOSPHOLIPIDS AND THEIR DEGRADING ENZYME, PHOSPHOLIPASE A2, IN HUMAN TEARS

Abstract

Abstract: : Purpose: Recent studies have shown that phospholipids are the most important class of lipids in maintaining stability of the tear film. The concentration of secretory phospholipase A2 (sPLA2), the enzyme that hydrolyzes phospholipids, in tears is known to exceed the levels found in serum by four orders of magnitude. We developed a sensitive method for measuring the amount of phospholipids and sPLA2 in human tears. Methods: Nineteen patients with dry eye, 12 soft contact lens wearers, and 18 normal subjects were enrolled in the study. All subjects gave informed consent for participation in the study. Unstimulated tears were collected with a Schirmer’s test strip in one eye of each subject. The lipid fraction was extracted from the tear sample by the method of Folch. Total lipids were determined by and sulfo–phospho–vanillin reaction. Phospholipids were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion of phospholipase C and alkaline phosphatase . Total protein was determined by BCA analysis, and sPLA2 activity in was measured by the use of 1, 2–diheptanoyl thio–phosphatidylcholine as a substrate. The concentration of type IIA sPLA2 was measured by ELISA. Results: Patients with dry eye and soft contact wearers had significantly lower amounts of phospholipids (218.6+/–183.3mg/L and 267.0 +/–188.1mg/L, respectively) in tears compared to normal subjects (432.1+/–225.1 mg/L), whereas concentrations of total lipids in tears did not differ among three groups. There was no statistically significant difference in sPLA2 activities or concentrations of type IIA sPLA2 in tears among the soft contact wearers, patients with dry eye and the normal subjects. Conclusions: The method used in this study enabled us to determine the quantities of lipids, phospholipids and their degrading enzyme in a small volume of tears. Deficiency of phospholipids, an important structural component of the polar phase of the tear film lipid layer, may be involved in the pathogenesis of dry eye syndrome and intolerant contact lens wearers.

Keywords: cornea: tears/tear film/dry eye • lipids • contact lens