Determination of Collagen Crosslinks in Rockfish Skeletal Muscle

The low collagen content in fish muscle makes the quantitative analysis of collagen crosslinks difficult. We have developed a method for the quantitative determination of reducible difunctional collagen crosslinks in samples containing as little as 0.5 picomoles of crosslink per microgram of hydroxyproline. The low collagen-containing tissue is first enriched by removing most of the non-collagenous protein with cold sodium hydroxide solution. The lyophilized alkali-insoluble extract is then reduced with NaB3H4 and acid-hydrolyzed. Initial fractionation of hydrolysates on a Bio-Gel P-2 gel filtration column provides partial separation of components with a sharp peak of radioactivity containing more than 90% of the difunctional crosslinks. This peak is ultimately analyzed by HPLC for the quantitative determination of specific difunctional crosslinks. This method has been used to show that the difunctional crosslink composition of muscle collagen from two species of Pacific rockfish differs quantitatively from each other and from that of rat tail tendon.