Measurement of Human Tear Lysozyme Using a Novel Synthetic Substrate

Lysozyme is a bacteriolytic enzyme representing approximately 30% of the protein content of human tears.1 Human tear lysozyme production generally parallels aqueous tear secretion and is a good indicator of lacrimal gland function. However, in some instances of ocular pathology, tear lysozyme concentration (HTL) may be decreased before aqueous tear production is reduced.2 Various spectrophotometric, turbidimetric, and immunologic methods for quantitating HTL have been proposed. The most common methods for measuring HTL are based on the ability of lysozyme to cleave the cell wall of Micrococcus lysodeikticus. These techniques suffer from poor standardization, variable results, and the need for specialized personnel to maintain reagents or operate equipment. The purpose of this study was to utilize a known biochemical reaction based on the selective action of lysozyme on N-acetyl-oligosaccharides to develop a rapid, reliable, and sensitive colorimetric methods for the measurement of HTL. The chemical substrate selected for this assay was p-nirrophenyl penta-N-acetyl B-chitopentaoside (PNP).3 The reaction of lysozyme with this substrate releases p-nitrophenyl N-acetyl B-D-glucosaminide, which can be coupled to a reaction with B-N-acetylhexosaminidase (NAHase) to liberate p-nitrophenol. The free p-nitrophenol can then be accurately quantitated with a simple colorimeter.