Title
Pre-column derivatisation method for the measurement of glycosylated hydroxylysines of collagenous proteins
Author
Bank, R.A.
Beekman, B.
Tenni, R.
Tekoppele, J.M.
Gaubius Instituut TNO
Publication year
1997
Abstract
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
Subject
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
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DOI
https://doi.org/10.1016/s0378-4347(97)00392-7
TNO identifier
234270
ISSN
1572-6495
Source
Journal of Chromatography B: Biomedical Applications, 703 (1-2), 267-272
Document type
article