Free Aldehydic Groups in Collagen and Other Tissue Components

Abstract

THE reaction of free carbonyl groups with N-methyl benzothiazolone hydrazone (MBTH) is a very sensitive and specific method for the quantitative determination of free aldehydes^1,2. Carbonyl compounds react with 1 mole of MBTH to form an azine derivative. Azines of aldehydes react sequentially with a second mole of MBTH, oxidized by ferric ion under acid conditions, to form blue-green tetraazopentamethine cyanine dyes³. These products can be specifically identified for aldehydes by their absorption spectra^2,4–6. While a variety of compounds will form azines with 1 mole of MBTH, the second stage of the reaction is specific for aldehydes and can be carried on in the presence of other carbonyl compounds. Carbohydrates having pyranose structures do not react with MBTH, nor do they interfere with the reaction of aldehydes with MBTH. The reaction is at least 100 times more sensitive than the Schiff reaction⁷. It differs from the periodic-acid–Schiff (PAS) reaction⁸ in that the MBTH reaction detects ‘natively’ free aldehydic groups while the PAS reaction uncovers the aldehydic group through oxidative reactions prior to reaction with Schiff's reagent.