Development of a chitin assay for the quantification of fungus

Chitin, a unique structural polysaccharide found in fungi and arthropods, is not produced by vertebrates. Thus, the potential applications of a specific and sensitive assay for chitin are numerous, including the evaluation of the extent of fungal keratitis. Chitin is a homopolymer of beta (1, 4) linked D-N-acetylglucosamine. We have developed a simple and reproducible assay for chitin and applied it to Candida albicans cultures. The assay involves homogenization of the culture and treatment with 21.1 M KOH to remove soluble materials, including proteins. This base treatment also deacetylates the chitin to the glucosamine polymer, chitosan. Chitosan is hydrolyzed by 0.5 M H2SO4 to glucosamine monomers which are then deaminated by the addition of NaNO2 to the acid solution. The resulting 2,5-anhydromannose is reduced by NaB[3H]4 to 1-[3H] 2,5-anhydromannitol. This radiolabelled sugar is isolated by paper chromatography and quantified via liquid scintillation. The sensitivity of this assay is assessed by comparison of colony forming units (CFU's) with a glucosamine standard. A typical run of the assay detects 53.1 CFU/c.p.m., and 356,000 c.p.m. per nanomole of N-acetylglucosamine. The specificity of the assay is very high because of the unique nature of chitin. This method of chitin determination may be a useful alternative method for future investigations involving the study of fungal infections in mammalian tissues.