A SIMPLIFIED METHOD FOR THE ANALYSIS OF HYDROXYPROLINE IN BIOLOGICAL TISSUES

A critical study of the different steps involved in previous procedure for hydroxyproline assay allows the direct measurement of collagen co ntent in tissue homogenates without losing the advantages of the metho d. The procedure is based on alkaline hydrolysis of the tissue homogen ate and subsequent determination of the free hydroxyproline in hydroly zates. Chloramine-T was used to oxidize the free hydroxyproline for th e production of a pyrrole, The addition of Ehrlich's reagent resulted in the formation of a chromophore that can be measured at 550 nm, Opti mal assay conditions were determined using tissue homogenate and purif ied acid soluble collagen along with standard hydroxyproline. Critical parameters such as the amount of chloramine-T, sodium hydroxide, p-di methylaminobenzaldehyde, pH of the reaction buffer, and length of oxid ation time were examined to obtain satisfactory results, The method ha s been applied to samples of tissue homogenate and purified acid solub le collagen, with recovery of added hydroxyproline of 101 +/- 6.5 and 104 +/- 6.0 (SD) percent, respectively. The method is highly sensitive and reproducible when used to measure the imino acid in tissue homoge nates. The modified hydroxyproline assay presented in this communicati on will be useful for routine measurement of collagen content in extra cts of various tissue specimens. In addition, the modified method can be used for batch processing of column fractions to monitor the collag en concentrations during purification.