Exp. Eyu Res.(1990) 50. 45-50 Sialic Acid in Human Tear Fluid 45 AB KUIZENGA*, ERIC J. van AGTMAAL, NICOLAAS J. van HAERINGEN AND AIZE KIJLSTRA” The Netherlands Ophthalmic Research Institute, Biochemical Laboratory, P.O. Box 72741, 7 700 AC Amsterdam, The Netherlands and =Department of Ophthalmology, University of Amsterdam, the Netherlands (Received 13 April 1989 and accepted in revised form 77 August 7989) A simple assay for the determination of sialic acid (N-acetylneuraminic acid) in human tear fluid was evaluated. Sialic acid, terminally bound on carbohydrate side-chains of glycoproteins, was releasedafter treatment with neuraminidase and measured by an enzymatic calorimetric test. Tear fluid sampleswere collected from ten healthy adults, using glass capillaries and cellulose sponges.Sialic acid levels in tears collected with sponges (08-1.8 mmol 1-l) did not differ significantly from those found in capillary tears (09-1.8 mmol 1-l). Sialic acid, expressed as mmol g-’ protein, was significantly lower in tears collected with sponges (018-0.32 mmol g-‘) than with capillaries (0.19-0.42 mmol g-l). Recovery of sialic acid and protein after incubation of cellulose sponges with tears was more than 99 %. Sialic acid levels in human tears, which had been centrifuged to remove insoluble material, remained unchanged. Furthermore, tear sialic acid activity did not pass a filter with a molecular weight cut-off point of 10000. Our data indicate that with the assay used in this report, sialic acid in tears is not due to secretory immunoglobulin A (sIgA), lactoferrin and lysozyme. The fact that the major tear proteins do not contribute to the sialic acid levels detectedin tears suggests that other as yet unknown soluble glycoproteins are involved. Key words: sialic acid: tear fluid; glycoproteins 1. Introduction The preocular tear film covers the cornea1 and conjunctival epithelia. Its functions are to maintain structural integrity of the cornea and conjunctiva and to protect the eye against external influences. The composition of human tear fluid is very complex (van Haeringen, 1981) and contains various sub- stances, including lipids, electrolytes, proteins and muco-glycoproteins. The understanding of the muco- glycoproteins on the ocular surface is gradually increasing. The mucous layer, covering the epithelial surface of the cornea and conjunctiva and containing mucous glycoproteins (mucins) (Moore and Tiiany, 1979) is considered to maintain the stability of and contribute to the viscosity of the tear film (Kaura and Tiffany, 1986). Mucous glycoproteins are macromolecules of vary- ing molecular weight and consist of a large linear protein core, to which many oligosaccharide side chains attach, containing hexoses and sialic acid. They can exist in water-soluble form and in an insoluble high molecular weight gel-form. The serum- type glycoproteins have a lower molecular weight and contain fewer carbohydrate side chains, attached to a more globular protein core. Although the conjunctival goblet cells are the main source for mucous glycoproteins (Kessing, 1968), glycoproteins have also been found in lacrimal gland acini (Allen, Wright and Reid, 1972) and glyco- * For correspondence. 00144835/90/010045+06 $03.00/O proteins in tears have been demonstrated by their hexosamine and sialic acid content (Cabezaset al., 1964; Dohlman et al., 1976), suggesting lacrimal secretion of these proteins onto the ocular surface (Kreuger, Sokoloff and Botelho, 1976). Water-soluble glycoproteins were detectedin tears by electrophoresis (Berta and Tiirak. 1986) and may be altered in various pathological states of the eye (Dohlman et al., 1976 ; Halken, Bog-Hansen and Prause, 1986a). Glycoproteins of lacrimal, together with those of conjunctival origin, can be important in maintaining the physicochemical properties of the precorneal tear film. Until now no rapid and simple method was available to quantitate glycoprotein levels in human tears. Therefore, the determination of sialic acid in human tear fluid, using a simple enzymatic colori- metric method, was evaluated as a parameter for tear glycoproteins. The major tear proteins do not contribute in the sialic acid assay used and it is concluded that sialic acid in human tears is mainly due to soluble glycoproteins of still unknown identity. 2. Materials and Methods Collection of Tear Fluid Tear samples were collected from healthy adults (n = 10) using 25+1 glass capillaries (capillary tears). Production of tear fluid was stimulated by a jet of pressurized air onto the cornea. Within a period of 15 min tear fluid production ranged from 25 ~1 to 0 1990 Academic Press Limited