Evaluation and modification of spectrophotometric procedures for analysis of lactate dehydrogenase, beta-glucuronidase and arylsulphatase in human gingival crevicular fluid collected with filter-paper strips

Filter-paper strips were used to collect GCF, and the sample eluted into a larger volume of diluent. This procedure allows for detection of site-to-site variation in GCF volume, and provides a 300–400 μl sample for analysis of lactate dehydrogenase (LDH), beta-glucuronidase (BG) and aryl-sulphatase (AS) activities by a standard (serum) spectrophotometric assay modified for increased sensitivity. The results indicate that although the standard assay for LDH (based on oxidation of NADH) was adequate for detecting low activity in GCF samples, the modification doubled the sensitivity and allowed the use of less sample volume, thereby providing additional material for other assays. The standard assay for BG based on phenolphthalein being generated from phenophthalein glucuronic acid was not adequate for use in GCF analysis. The modification used increased assay sensitivity five-fold and allowed smaller samples to be used. The serum assay for AS (conversion of nitrocatechol sulphate to nitrocatechol) was accurate to the lower limit of AS activity in GCF and could be used without modification. The results emphasize the need to evaluate critically standard spectrophotometric assays for sensitivity when studying physiologically-collected GCF.