Eine empfindliche Methode zur Bestimmung von Glykosaminoglykanverteilungsmustern, angewendet auf 9 Bandscheiben einer menschlichen Wirbelsäule

Abstract

Zusammenfassung: Es wird eine Methode zur Bestimmung des Glykosaminoglykanverteilungsmusters in einem isolierten Glykosaminoglykanpool beschrieben, die auf der aufeinanderfolgenden Anwendung der spezifischen Enzyme Hyaluronatlyase, Chondroitinsulfatlyase AC und ABC beruht. Die Bestimmung der Komponenten erfolgt empfindlich über die Metabolitkonzentrationen mithilfe der Hochleistungsflüssigkeitschromatographie (Nachweisgrenzen unter 0,1 nmol). Unverdautes Heparan-und Keratansulfat kann dünnschichtchromatographisch getrennt und mit Hilfe der Uronsäure-bzw. Hexosaminbestimmüng quantifiziert werden.Die mit dieser Methode ermittelten Verteilungsmuster stimmen nur dann mit denen'überein, die man mit Hilfe der lonenaustauschchromatographie (Dowex 1X2) erhält, wenn die Fraktionen enzymatisch charakterisiert werden. Die Anwendung der Methode auf 9 Bandscheiben aus einer menschlichen Wirbelsäule (Anulus fibrosus und Nucleus pulposus jeweils getrennt) ergab 1) keine systematische Veränderung des Verteihmgsmusters in Abhängigkeit von der Lage der Zwischenwirbelscheiben im mittleren Abschnitt der Wirbelsäule und 2) im Anulus ein im Vergleich zum Nucleus niedrigeres Verhältnis von Galaktosaminoglykan zu Keratansulfat. A sensitive method for the analysis of the glycosaminoglycan distribution pattern, applied to 9 intervertebral discs ofone human spineSummary: A method has been established for anälysing the glycosaminoglycan distribution pattern in small specimeris from intervertebral discs arid other connective tissue materials. The procedure is based on the consecutive digestion by the specific enzymes hyaluronate lyase, chondroitin sulfate lyase AC and ABC. The quantitative determination of the resülting disaccharides was achieved by high performance liquid chromatography with a detection limit of less than 0.1 nmol. The undigested heparan-and keratansulfate were separated from each other by thin layer chromatography and quantified by the eolorimetric determination of urpnic acid and hexosamine, respectively. Correlätiön öf distribution pattenis obtäined by this jnethod with those obtained by ion exchange chromatography (Dowex l X 2) is not ppssible without the additiqnal enzymatic characterization of the ion exchange Chromatographie fractions. The method was applied to 9 discs öf one human spine (anulus fibrosus and nucleus pulposus separately) . with the föliowirig results: 1) there were no systematic changes to be seen in the distribution pätterns with respect to the location of the discs in the spine, and

High Performance Liquid Chromatographie Assay of Disaccharides and Oligosaccharides Produced by the Digestion of Glycosaminoglycans with Chondroitin Sulphate Lyases

“…After chondroitinase ABC degradation, the undigested residue was separated by thin-layer chromatography and keratan sulphate and heparan sulphate were quantified by determining hexosamine (18) and uronic acid (17) respectively, s described in a previous publication (6). The columns were protected by using 20 χ 4 mm cartridges s precolumns, prepacked with Nucleosil 5 SB (Bischof Analysentechnik, Leonberg, FRG).…”

“…The method used to isolate the glycosaminoglycans from human intervertebral discs, articular eartilage and Dupuytren's contracture has been described in füll elsewhere (6). Briefly, the tissues were subjected to the following procedures: proteolysis with papain, precipitation of non-glycosaminpglycan material with perchloric acid at pH 1.3, dialysis against double-distilled water, freeze-dyring, ß^elimination in 0.…”

“…The specific enzymes hyaluronate lyase, chondroitinase AC and Chondroitinase ABC are being used increasingly for the determination of individual glycosaminoglycan components (1)(2)(3)(4)(5)(6)(7). These enzymes digest hyaluronate, chondroitin sulphate and dermatan sulphate to form -ß-unsaturated uronic acids.…”

“…Sequential application of these enzymes in the above order, and Separation of the metabolites from the undigested glycosaminoglycans after each digestion Step permits the determination of glycosaminoglycan distribution patterns. Quantification may then be performed by analysing the metabolites of the digested components (6) or by determining the concentrations of the undigested glycosaminoglycans (5,7). Metabolite analysis is the preferred method because this also yields Information on the degree of sulphation and the proportion of 4-sulphated and 6-sulphated isomers.…”

“…One analytical procedure for the determination of glycosaminoglycan distribution patterns has been reported which is based on the sequential application of hyaluronate lyase, chrondroitinase AC and chondroitinase ABC and high performance liquid Chromatographie analysis of the products of degradation (6). This procedure has been used to identify glycosaminoglycan distribution patterns in various human connective tissues.…”