PRECOLUMN DERIVATIZATION METHOD FOR THE MEASUREMENT OF GLYCOSYLATED HYDROXYLYSINES OF COLLAGENOUS PROTEINS

Measurement of the glycosylated hydroxylysines galactosyl-and glucosyl galactosylhydroxylysine (GH and GGH) in combination with other amino a cids has been based on ion-exchange chromatography followed by reactio n with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic method with fluorimetric detection has been developed and employed to determine the glycosylated hydroxylysine residues in a lkaline collagen hydrolysates. After hydrolysis, amino acids were deri vatised with 9-fluorenylmethyl chloroformate and separated on a Microp ak ODS-80TM reversed-phase column (150x4.6 mm). With a multistep gradi ent system all amino acids were separated in less than 30 min, includi ng the collagen-specific hydroxylysine, hydroxyproline and the glycosy lated hydroxylysines. The method was used to evaluate the glycosylatio n levels of human articular cartilage derived from femoral head, femor al condyle, tibial plateau and ankle. GGH was highest in cartilage fro m femoral head and ankle; GH showed no differences between the differe nt sources of cartilage. (C) 1997 Elsevier Science B.V.