desmosine and phenylisothiocyanate

A method has been developed for the separation and quantitation of desmosines in tissue samples. The tissue is treated with cold 10% trichloroacetic acid to remove collagen and hydrolysed in HCl vapours in sealed vials. Preseparation of desmosines from tissue acid hydrolysates is performed on a cellulose column, first eluted with n-butanol-acetic acid-water to wash out other amino acids and then with water to recover desmosines. Separated desmosines are then derivatized with phenylisothiocyanate and determined by reversed-phase high-performance liquid chromatography using a gradient system with sodium acetate pH 6.4 and acetonitrile. Desmosines were detected spectrophotometrically at 254 nm. The method was applied to the determination of desmosine in elastin, rat aorta and bovine ligamentum nuchae.

Topics: Animals; Aorta; Cattle; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Desmosine; Elastin; Isothiocyanates; Ligaments; Rats; Spectrophotometry, Ultraviolet; Thiocyanates

A new sensitive and selective high-performance liquid chromatographic method for the analysis of desmosine and isodesmosine in human and rat tissues is described. This method requires a purification step with column chromatography, followed by precolumn derivatization phenylisothiocyanate. The reaction products are then separated by isocratic chromatography on a C18 column and quantitated by ultraviolet detection at 254 nm. The recovery of standards of both compounds added to tissue samples and analysed by this method is usually greater than 90%, and the absolute detection limit is 0.5 ng for both compounds. The method is sensitive enough to measure both substances in tissue fragments of 30 mg of wet mass, which means that it can be used to study elastin in small human biopsies.

Topics: Animals; Aorta; Chromatography, High Pressure Liquid; Desmosine; Humans; Isodesmosine; Isothiocyanates; Lung; Male; Rats; Spectrophotometry, Ultraviolet; Thiocyanates; Veins