Hydroxyproline determination in serum and gingival crevicular fluid

Abstract

A high‐performance liquid chromatographic assay for total hydroxyproline in serum and gingival crevicular fluid has been developed. The Clq component of the first complement factor was precipitated out of the samples with a 0.02 M sodium acetate solution. Aliquots of the supernatants were dried, vapor hydrolyzed with 6 M hydrochloric acid at 105°C for 24 hours and derivatized with phenylisothiocyanate. The derivatives were chromatographed using a multilinear solvent gradient with detection at 254 nm. The regression of peak areas on the concentrations of hydroxyproline was linear with a correlation coefficient of 0.99 within the range of assayed quantities (2.5 to 25 ng). Analytical recovery was 90.5 ± 2.54% (X̄± SD) and the determination level was 2.5 ng (19.1 pmol). The precision of the assay as determined by the coefficient of variation for five consecutive runs of six concentrations of hydroxyproline in serum hydrolysate (“within‐run” precision) and five series run on different days (“between‐run” precision) was 1.8 ± 1.28% and 4.2 ± 2.59% (X̄± SD), respectively. Hydroxyproline concentrations in GCF from single sites during developing experimental gingivitis in the beagle dog showed an irregular pattern of low and high concentrations ranging from 5.2 to 17.4 ng/μl.