Measuring procollagen and collagen type I and III kinetics using stable isotope techniques

Collagen synthesis is one of the major processes in skin regeneration and wound healing. Efforts have been made to define collagen synthesis and breakdown to quantify wound healing, however, specific quantification of wound repair rates have been difficult. In this study, we developed a method of measuring skin collagen synthesis and breakdown using a primed-constant infusion of 1,2-¹³C-Glycine. Blood and skin sampling of male Sprague-Dawley rats were taken as shown in the following study design: (Figure Presented) Skin was homogenized in an extraction buffer and collagenous proteins precipitated with saturated ammonium sulfate. Collagen types I and III were separated by salt precipitation, and after dialysis the samples were purified and separated into procollagen and collagen tractions on a DEAE cellulose column. The samples were lyophilized, hydrolyzed, purified and derivatized using t-BDMS. Glycine enrichment was determined by gas chromatography/mass spectrometry and the fractional synthetic rate and the fractional breakdown rate of collagen were calculated. This method will allow quantification of collagen kinetics in wound healing.