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Sensitive fluorimetric quantitation of pyridinium and pentosidine crosslinks in biological samples in a single high-performance liquid chromatographic run

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Author: Bank, R.A. · Beekman, B. · Verzijl, N. · Roos, J.A.D.M.de · Nico Sakkee, A. · Tekoppele, J.M.
Type:article
Date:1997
Institution: TNO Preventie en Gezondheid
Source:Journal of Chromatography B: Biomedical Applications, 1-2, 703, 37-44
Identifier: 234268
doi: doi:/10.1016/S0378-4347(97)00391-5
Keywords: Biology · Biomedical Research · Collagen · Pentosidine · Pyridinium · collagen · pyridinium derivative · animal tissue · articular cartilage · bone · cattle · collagen synthesis · connective tissue · cross linking · high performance liquid chromatography · human tissue · measurement · nonhuman · priority journal · reproducibility · reversed phase high performance liquid chromatography · standard · urine · Adolescent · Arginine · Bone and Bones · Cartilage, Articular · Chromatography, High Pressure Liquid · Circadian Rhythm · Connective Tissue · Cross-Linking Reagents · Humans · Hydrolysis · Ligaments · Linear Models · Lysine · Middle Aged · Pyridinium Compounds · Reproducibility of Results · Sensitivity and Specificity · Spectrometry, Fluorescence · Tendons

Abstract

A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 μm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra- assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well. Chemicals/CAS: Arginine, 74-79-3; Cross-Linking Reagents; Lysine, 56-87-1; pentosidine, 124505-87-9; Pyridinium Compounds