An agarose gel matrix was utilized to grow chondrocytes from human donors of various ages in cell culture. The chondrocytes produced the pericellular matrix characteristic for such cells and synthesized collagen type II as well as glyco-saminoglycans. The latter exhibit the typical distribution pattern of the respective articular cartilage matrix. The electron-microscopic appearance of the cultured chondrocytes closely resembles that of chondrocytes in sections of the original cartilage.

Gurr et al.: HPLC assay of disaccharides and oligosaccharides 77 Summary: In high perfonnance liquid Chromatographie procedures hitherto described, SiO 2 , NH 2 and RP columns have been used for the analysis of disaccharides produced by % the digestion of glycosaminoglycans with the chondroitin sulphate lyases AC and ABC. The use of a potent anion exchanger offers the following advantages over these columns: superior Separation chäracteristics for non-sulphated disaccharides, and improved column perfonnance, coupled with more stable analytical conditions. Elution with dilute saline Solutions permits Separation of the two non-sulphated disaccharides from chondroitin and hyaluronate.The sequential application of chondroitinase AC and ABC permits the determination of hyaluronate, the chondroitin sulphate isomers and the dermatan sulphate isomers by high perfonnance liquid Chromatographie Separation of the products of enzymatic hydrolysis. In a previously described method, hyaluronate lyase was used for the determination of hyaluronate. It has been found, however, that omission of the hyaluronate lyase step results in superior accuracy in the high performance liquid Chromatographie Separation of the nonsulphated disaccharides.The enzymatic analysis of human articular cartilage glycosaminoglycans has repeatedly yielded a fraction which is not digestable by chondroitinase AC, but is completely digestable by chondroitinase ABC. More extensive characterization has disclosed that this fraction differs structurally from chondroitin sulphate. Enzymatic characterization indicates that it should presumably be assigned to dermatan sulphate. Hochleistungsflüssigkeitschromatographische Bestimmung von Di-und Oligosacchariden aus dem Abbau von Glykosaminoglykanen mit ChondroitinsulfatlyasenZusaHunenfassung: Bei den bisher beschriebenen hochleistungsflüssigkeitschromatographischen Verfahren zur Analyse von Disacchariden aus dem Abbau von Glykosaminoglykanen mit den Chondroitinsulfatlyasen AC und ABC wurden SiO 2 -, NHk-und RP-Säulen verwendet. Gegenüber diesen Säulen bietet die Verwendung eines starken Anionenaustauschers folgende Vorteile: bessere Trenneigenschaften im Bereich der unsulfatierten Disaccharide und höhere Laufleistung pro Säulenfällung bei stabileren Analysenbedingungen. Die Elution mit verdünnten Kochsalzlösungen ermöglicht die Trennung der beiden unsulfatierten Disaccharide aus Chondroitin und Hyaluronat.Durch die sequentielle Anwendung von Chondroitinase AC und ABC können durch hochleistungsflüssigkeits-chromatographische Trennung der Metabolite Hyaluronat, die Chondroitinsulfatisomere sowie die Dermatansulfatisomere bestimmt werden. Verglichen mit einer früher beschriebenen Methode, bei der zur Hyaluronatbestimmung Hyaluronatlyase verwendet wurde, zeigt sich, daß ohne den Hyaluronatlyase-Schritt durch hochleistungsflüssigkeitschromatographische Trennung der unsulfatierten Disaccharide eine bessere Richtigkeit erreicht wird.

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

Eighty specimens from 20 patients with Dupuytren's disease and 7 biopsies of healthy palmar fascia were analysed for their glycosaminoglycan isomer patterns with a combined enzymatic/HPLC method. The diseased portions of palmar fascia tissue were characterized by elevated total glycosaminoglycans together with a relative increase in the sulphated fractions. The macroscopic stages of nodules, bands and unaffected tissue could be classified very well by multivariate statistical analysis on the basis of their glycosaminoglycan patterns. The biochemical analysis provided evidence of the pathological process even in those specimens that did not yet show any clinical symptoms of the disease.Because of the role played by sulphated glycosamiThe mechanisms involved in the pathogenesis of Du-noglycans in the interaction with collagen (12), the puytren's disease are still unknown (1). Recent studies question arises of whether there are changes in the on the morphological changes in diseased tissue have proportions of glycosaminoglycans and their unsuldemonstfated occlusioii of capillaries by cells that phated and sulphated isomers in affected palmar fasmigrate into the perivascular space. These cells are cia that may have an influence on the collagen pattern thought to be the origin of dedifferentiated cells that and the macromolecular structure. The present study proliferate and metabolize in such Way that they cause was conducted to determine the types and amounts contracture of the aponeurosis (2, 3). As to the pa-of the various sulphated and unsulphated glycosathobiochemistry, an increase in collagen type III pro-minoglycans and to reassess the data published so far tein has been reported (4-7), as has an increase in on the glycosaminoglycan content of both healthy the proportion of chondroitin sulphate (4, 8 -11). human palmar fascia and tissue from Dupuytren'ŝ contracture, by employing a method of determination ') With support of the Deutsche Forschungsgemeinschaft (SFB more specific than any used previously. The new 54). method also enabled differentiation of the unsul-2 ) Present address: Institute for Clinical Chemistry and Labo-, , ~A . . -, n< e "i«i^t-/i ^mnr^ntc ratory Medicine, university Hospital "BergmannsheiT Bo-P hated > C4-sulphated and C6-sulphated components chum, FRG (11).

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