THE DETERMINATION OF COLLAGEN AND ELASTIN IN TISSUES

Abstract

In the study of the occurrence and distribution of collagen in normal and pathological tissue and its formation and development in the embryo there has been need for aq accurate method to determine this substance in small amounts of tissue. Spencer, Morgulis, and Wilder (1) devised a method based on conversion of collagen to gelatin followed by the determination of the nitrogen of the gelatin precipitated with tannic acid. There is no assurance that only gelatin is extracted and precipitated under the given conditions. Later work (2) has indicated that excess nitrogenous material is measured by this method. Lowry, Gilligan, and Katersky (2) developed a gravimetric procedure involving extraction of soluble non-collagenous substances with 0.1 N NaOH at room temperature and conversion of collagen to gelatin by autoclaving; collagen was determined by difference in the dry weight of the residues. It was necessary to assume that different tissues behaved alike under the standard conditions and that the various fractionating procedures specifically effected the separation of the different components of the tissues. Abercrombie and Johnson (3) obtained an extract of nerve tissue by autoclaving according to the procedure of Lowry el al. (2) and calculated collagen from the nitrogen content of the extract. Elastin has been determined by weighing the fraction of tissue resistant to solution in 0;l N NaOH at 100” (2). A simple method is described here for determining collagen in small amounts of tissue with a precision and accuracy usually better than f5 per cent. In tissues containing but little collagen much of the soluble protein is separated from the tissues with the aid of 20 per cent urea solution. (Tissues containing large amounts of collagen do not require this preliminary separation.) The collagen is converted to soluble form (gelatin) by hydrolysis with water in an autoclave and thus extracted from elastin and other substances not dissolved by the process. The collagen content is estimated from the hydroxyproline content of acid hydrolysates of the extract.