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Pre-column derivatisation method for the measurement of glycosylated hydroxylysines of collagenous proteins

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Author: Bank, R.A. · Beekman, B. · Tenni, R. · Tekoppele, J.M.
Institution: Gaubius Instituut TNO
Source:Journal of Chromatography B: Biomedical Applications, 1-2, 703, 267-272
Identifier: 234270
doi: doi:/10.1016/S0378-4347(97)00392-7
Keywords: Biology · Biomedical Research · Collagen · Derivatisation, LC · Galactosylhydroxylysine · Glucosylgalactosylhydroxyl ysine · Hydroxylysines · collagen · hydroxylysine · amino acid analysis · ankle · articular cartilage · controlled study · femur condyle · femur head · human tissue · ion exchange chromatography · priority journal · protein glycosylation · reversed phase high performance liquid chromatography · tibia · Animals · Ankle · Cartilage · Cartilage, Articular · Cattle · Chromatography, High Pressure Liquid · Collagen · Female · Glycosylation · Hip · Humans · Hydrolysis · Hydroxylysine · Knee · Middle Aged · Nose · Placenta · Pregnancy · Sensitivity and Specificity · Spectrometry, Fluorescence · Time Factors


Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction 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 alkaline collagen hydrolysates. After hydrolysis, amino acids were derivatised with 9- fluorenylmethyl chloroformate and separated on a Micropak ODS-80TM reversed- phase column (150 x 4.6 mm). With a multistep gradient system all amino acids were separated in less than 30 min, including the collagen-specific hydroxylysine, hydroxyproline and the glycosylated hydroxylysines. The method was used to evaluate the glycosylation levels of human articular cartilage derived from femoral head, femoral condyle, tibial plateau and ankle. GGH was highest in cartilage from femoral head and ankle. GH showed no differences between the different sources of cartilage. Chemicals/CAS: Collagen, 9007-34-5; Hydroxylysine, 28902-93-4