טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTsitron Eve
SubjectMolecular Age and Turnover of Proteoglycans and
Collagen of the Human Intervertebral Disc
DepartmentDepartment of Biomedical Engineering
Supervisors Professor Alice Maroudas
Ms. Sarit Sivan


Abstract

Human intervertebral disc (IVD) is the largest avascular cartilaginous structure in the body. Its mechanical function is performed by a highly organized matrix whose major components are collagen and aggrecan. During aging and degeneration, structural and biochemical changes occur in the proportions and properties of these two constituents, especially that of aggrecan. These changes include an increase in the proportion of lower molecular weight aggrecan monomers and of the "free" binding region. One of the unresolved questions is whether the changes in the two components are due to alterations in the existing molecular structures or to changes in the newly synthesized species. Clearly, the residence time of the molecules would be very different in each of these cases. An important question is therefore how rapid is their turnover. Aspartic acid is one of the most rapidly racemizing amino acids, thus allowing quantitation of the concentration of D-isomers in long-lived proteins during a human life span. By measuring the D/L-aspartic acid ratio of the different aggrecan species as a function of age we were able to establish directly the relative residence time of these molecules in human IVD matrix. For the large aggrecan monomer, the turnover is 0.13±0.042 per year, corresponding to a half life of 5.56±1.58 years; whereas the turnover for the binding region is 0.033±0.0012 per year corresponding to half life of 21.53±0.6 years. Moreover, for mixture of monomers, the following mean half-life values of 12.0±2.01 and 11.23±1.87 years were found for normal nucleus pulposus and annulus fibrosus respectively, and 8.77±2.169 and 8.42±2.84 for degenerate nucleus pulposus and annulus fibrosus respectively. This difference may be due to increased synthesis of the intact monomer in the degenerate tissue or to an increased rate of removal of small aggrecan fragments from the tissue. For collagen, the mean half-life values of 131.3±18.5 and 122.8±18.4 years were found for nucleus pulposus and annulus fibrosus respectively. Thus, it can be concluded that virtually no turnover occurs during the lifespan of an individual. Pentosidine is a cross-link between lysine, sugar and arginine, formed as a consequence of a non-enzymatic glycation in proteins. Pentosidine formation rate (kF) was determined by using data of pentosidine accumulation over the years and turnover rate of the proteins, kt , obtained from the aspartic acid analyses. Pentosidine formation of collagen and aggrecan were found to be similar, suggesting that turnover rate has greater impact compared to formation rate on the accumulation of pentosidine.