TRANSFORMATIONS OF RAT SKIN COLLAGEN WITH SPECIAL REFERENCE TO THE AGEING PROCESS
Authors: HEIKKINEN EINO 
Published: December 2008

SummaryThe formation of insoluble collagen in rat skin was studied both invivoandin vitroin order to clarify the mechanism by which tropocollagen units are transformed into supermolecular structures, especially in relation to the ageing process.I. Suitable methods were developed for the fractionation and purification of soluble collagens (Fig. l), for the determination of the specific radioactivity of hydroxyproline in starch‐gel electrophoretic collagen fractions (Table IV, Fig. 2) and for the production of insoluble collagenin vitro(Fig. 12).II. The proportion of insoluble collagen was maximal in the skin of a newborn rat. The rapid growth period of the rat was characterized by an accumulation of collagen soluble in 0.5Macetic acid at 4° (Fig. 3). The insoluble collagen was separated into two subfractions (designated ISC40and ISCr). The proportion of collagen soluble in 0.5Macetic acid upon heating at 40° (ISC40) increased with age (Figs. 4–5). ISCRcould be brought into solution only after degradation of the collagen at temperatures above 60° (Fig. 5). Also the proportion of ISCRwas maximal in the skin of a newborn rat (Fig. 4).The subfractions of insoluble collagen extracted by 0.5Macetic acid at 40° from skins of 5‐day‐old rats consisted mainly of α‐ and β‐components. The proportions of β‐ and γ‐components were higher in corresponding fractions from skins of adult rats (Fig. 7). The ratio of hexmamine to hydroxyproline decreased rapidly in the insoluble collagen with advancing age (Table V).The content of free aldehyde groups was lower in the collagen of a lathyritic rat than in the collagen of a normal rat. The difference was largest in the a‐components (Table VII). The subfractions of insoluble collagen had different contents of aldehyde groups: ISC60°‐80°contained one acetaldehyde equivalent/TC less than ISC20°‐50°(Table VI).Incorporation studies indicated that insoluble collagen is formed faster in young than in adult rats (Table IX, Fig. 9). The half‐lives of 0.5MNaC1‐soluble collagens from 6‐day‐old and 3‐month‐old rats were 27 and 73 hours, respectively (Fig. 10). The turnover of the α2‐component in 0.1Macetic acid‐soluble collagen from a 6‐day‐old rat was faster than that of the α1‐component (Fig. 11).III. When skin slices labelledin vivowere incubatedin vitro, the total and specific radioactivities of the hydroxyproline of the insoluble collagen increased during 8 hours (Table X, Fig. 12). The formation of insoluble collagen during incubation was augmented by (1) a rise in temperature of the incubation medium up to 37° (Fig. 13); (2) a decrease in the thickness of the slices to 0.3 mm (Table XI); and (3) the presence of calcium ions up to 3.0 mMconcentration (Fig. 15). The45Ca‐activity increased in the insoluble residue of skin when skin slices labelled invivowere incubatedin vitro(Table XIII).Metal cations influenced the formation of insoluble collagen in the following order of decreasing effectiveness (Table XIV and XV): Co2+> Cu2+> Cu+> Au3+= Ni2+> Fe3+> Hg2+> Cr3+> Ca2+> Fe2+> AI3+> Bb4+> Sn4+> Sr2+= Ba2+> Mn2+= Mg2+.The addition of EDTA and substances reacting with aldehyde groups (phenylhydrazine and semicarbazide) to the incubation medium (Figs. 16 ‐17) inhibited the formation of insoluble collagen.The rate of formation of insoluble collagen was lowest near pH 6.4 and increased in both alkaline and acid directions (Fig. 14).Attempts to influence the formation of insoluble collagenin vitroby employing anaerobic conditions and by adding sodium fluoride, sodium iodoacetate, αα‐dipyridyl and puromycin failed.The aldehyde groups in the 0.45MNaC1‐soluble collagen increased when skin slices were incubated in the presence of glucosein vitro(Table XX).Insoluble collagen was formed also when skin homogenates were incubatedin vitro(Table XVII).Two possible age‐dependent mechanisms leading to the formation of insoluble collagen are suggested. The roles of metal cations and aldehyde groups in the stabilization of collagenous structures are discussed.