| Research Abstract |
Collagen and elastin are recognized as two major connective tissue proteins of human connective tissues. In both collagen and elastin there are many kinds of intra- or inter-molecular crosslinks. Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are mature crosslinks which maintain the structure of the collagen fibril. Desmosine (Des) and isodesmosine (Isodes) represent the major crosslinking components of elastin. Pentosidine (Pen), which is a senescent crosslink and one of the advanced glycation endproducts, accumulates with age in tissue proteins including collagen. We developed a direct and one-injection HPLC method to measure Pyr, Dpyr, Des, Isodes and Pen in the hydrolysate of human yellow ligament. This method used one column and two detectors. Recovery rates of Pyr, Dpyr, Pen, Des and Isodes were 86.4-98.3%, 83.6-96.8%, 78.7-95.6%, 83.6-97.9% and 85.6-99.3% respectively. The intraassay coefficients of variation for Pyr, Dpyr, Pen, Des and Isodes were 3.7%, 4.1%, 5.4%, 4.5%, and 4
… More
.7% respectively. Linear regression analysis showed the linearity (r=0. 99, p=0. 0001) of calibration line for each Pyr, Dpyr, Pen, Des and Isodes. Using this HPLC method, we further tried to measure semi-quantitatively the content of collagen and elastin in connective tissues. Part of the connective tissues such as cortical bone, cancellous bone, cartilage, vertebral disc, meniscus, ligament, tendon, joint capsule, synovium, nerve, and skin were obtained. After hydrolysis of the samples, we measured crosslinks of collagen and elastin such as pyridinoline, deoxypyridinoline, desmosine, isodesmosine by HPLC at once. For calculating the contents of collagen and elastin, we estimated that 1 mole of collagen contains 0.2 moles of pyridinoline in bone ; 1.5 moles in cartilage, meniscus and disc, 0.4 moles in capsule, tendon, synovium, ligament and nerve, and 1 mole of elastin contains 3 moles of desmosine. In the ligament, capsule, synovium, and nerve, both collagen and elastin crosslinks were detected and thus the contents of collagen and elastin were semiquantified. The elastin crosslinks were not measured in the bone, cartilage, meniscus, disc, or tendon. The collagen crosslinks were not measured in the skin. Capsule had more collagen and less elastin, and the ligament had more elastin and less collagen. In the capsule, an old subject had the greater proportion of collagen to elastin than a young subject. We believe that this semi-quntitative HPLC method is useful for the investigation of the connective tissues which have significant amount of collagen and elastin. Less
|
