A method is described for the determination of plasma and serum glycosaminoglycans, which can be used in any laboratory equipped with an HPLC system. It is based on the sequential application of chondroitinases AC and ABC and separation of the resulting disaccharides by high-performance liquid chromatography. All reagents are commercially available. This simple and rapid separation yields an accurate quantification and an exact distribution pattern. The determination of glycosaminoglycan disaccharides is linear between 7 and 7000 μιηοΐ/ΐ with coefficients of variation between 3.0 and 7.7% for serum and between 2 and 14% for plasma. The recovery of the assay ranged from 93 to 106% for different concentrations of glycosaminoglycan disaccharides. This HPLC method may therefore be considered as a candidate reference method.
In each of 30 skeletally mature sheep, the posterior cruciate ligament was replaced in one knee by a free patellar tendon autograft using the central third of the ipsilateral patellar tendon. The healing autograft was compared with the contralateral posterior cruciate ligament and the patellar tendons and posterior cruciate ligaments of nonoperated animals. The content of glycosaminoglycans, chondroitin sulfate disaccharides, and dermatan sulfate disaccharides was assessed biochemically at six periods during the 2 years after surgery. The total glycosaminoglycans and chondroitin sulfate disaccharides in the native posterior cruciate ligament was threefold that in the native patellar tendon. In contrast, the amount of dermatan sulfate disaccharides was similar in both the native tendon and native ligament. In the autograft, glycosaminoglycans and chondroitin sulfate disaccharides increased significantly to about 144% and 172%, respectively, of the contralateral posterior cruciate ligament at Week 104. The dermatan sulfate disaccharides in the autograft also showed a significant increase up to Week 26, followed by a remarkable but not significant decrease until the end of the study. In the contralateral posterior cruciate ligament, the dermatan sulfate disaccharides increased significantly between Weeks 52 and 104. Thus, the amount of dermatan sulfate disaccharides was similar in both the autograft and the contralateral posterior cruciate ligament after 2 years. This study suggests that the patellar tendon autograft did not completely assume the biochemical properties of the posterior cruciate ligament.
Ausgehend von kiullichem 3-Methoxybeddehyd (I) wird rnit dem (4S.SS)-( +~S-Amin~~Z-dimcihyl-4-phenyl-1,3-dioxao (2)sls chitalm Hilfsamio und 3llausiure das enantimerenrehe t ( +)-Forphenicin (12) synthetisiert. U ist identiscb mit dem Naturprodukt, das aus der KuItwfliigsigkeit ehes Stammes von A c tinonryces isoliert wurde uod ais starker Inhibitor der alkdkchen Phosphatase beschrieben wird. L-( + )-Forphenicin (12) ist eine biologisch wichtige nichtproteinogene Aminosaure, die aus der Kulturfliissigkeit eines Stammes von Actinomyces isoliert wurde und ein starker Inhibitor der alkalischen Phosphatase ist 2,3). NaBH,Reduktion von 12 fiihrt zum sogenannten L-( +)-Forphenicinol (lo), das auf zwei Wegen aus dem 3-Hydroxy-4-hydroxymethyl-3,4-O-isopropylidenbenzaldehyd und 3-Hydroxybenzaldehyd in racemischer Form synthetisiert wurde4z5). Beide Synthesen erfordern am Ende eine Racematspaltung, um zu dem enantiomerenreinen L-( + )-Forphenicinol (10) zu gelangen, und sind daher unwirtschaftlich. Obwohl wir den 3-Hydroxy-4-hydroxymethyl-3,4-O-isopropylidenbenzaldehyd, der als farbloses 0 1 beschrieben wird4), kristallin herstellen konnten" und seine Reinheit analytisch und spektroskopisch bewiesen haben, lie13 er sich nicht mit (4S,5S)-( +)-5-Amino-2,2-dimethyl-4-phenyl-1,3-dioxan (2) zum kristallinen Aminonitril umsetzen. Ebenso konnte aus dem kauflichen 3-Hydroxybenzaldehyd das entsprechende Aminonitril nicht kristallin hergestellt werden. Damit sind beide Ausgangsverbindungen nach unseren friiheren Unter-suchungen7) fur die asymmetrische Synthese mit dem chiralen Hilfsamin 2 unbrauchbar.Durch die Synthese von enantiomerenreinem Anisylglycin und anderen methoxysubstituierten aromatischen Aminosauren9) ist bekannt, daD sich aus methoxysubstituierten aromatischen Aldehyden oder Ketonen mit 2 und Blausaure gut kristallisierende Aminonitrile herstellen lassen. Die Phenolether lassen sich am Ende der Synthese leicht und ohne Racemisierung der Aminosauren mit Bromwasserstoffsaure spalten. Aus dem enantiomerenreinen L-( + )-Ani~ylglycin~) ([~XI::~ = +175.6) kann auf diesem Weg das enantiomerenreine L-( + )-2-(4-Hydroxyphenyl)glycin ([a]:& = + 108.0) erhalten werden. Aus diesem Grund sind wir bei der Synthese von L-( +)-Forphenicin (12) vorn kauflichen 3-Methoxybenzaldehyd (1) ausgegangen.833 NwppDteinogeaic Amino Acids, IV'! -EPC Synthesis of L-( +> F Q -Enantiomerically pure 1-41. )-forphwicine (12) is synthesized starting from purchasable 3-methoxybenzaldehyde (1) by the use of (4S,SS).( + ~S-amino-2,2-dimethyl-4-phenyl-l,3-dioxa~e (2) as ac h i d auxiliary and prussic acid. I2 is identical with the natural product which has been isolated from the culture broth of an Actinomyces gpecies and which is known to be a potent inhibitor of alkaline phosphatase.
Summary: A competitive enzyme immunoassay was developed to determine chondroitin-6-sulphate in body fluids and cell cultures. The assay uses a monoclonal anti-chondroitin-6-sulphate antibody, immobilised to microtitre plates, and it involves a competitive binding reaction between chondroitih-6-sulphate in the samples and the biotinylated antigen.This assay enables the quantification of chondroitin-6-sulphate in the low concentration ränge of 16-120 g/l. The intra-assay and inter-assay coefficients of Variation are below 6.5% and 9.0%, respectively. More than 90% of chondroitin-6-sulphate was recovered when added to 0.1 mol/1 phosphate-buffered saline, an albumin solution (40 g/l in phosphäte-buffered saune) and cell culture medium (containing 100 ml/l foetal calf serum).Chondroitin-6-sulphate was also determined in sera of healthy male (n = 90) and female (n = 90) blood donors. The normal ränge was 55-169 g/l. In men the mean value was estimated at 102.2 ±37.1 g/l and in women at 98.7 i 26.4 g/l. Np age or sex dependence was observed.The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 ±21.1 mg/kg creatinine (mean ± Standard deviation) and in fernales (n = 10) 53.5 ± 21.3 mg/kg creatinine. The clearance rate in men was 0.41 ± 0.22 ml X min" 1 and in women €.38 ± 0.15 ml X min" 1 . No sex dependence was found.Furthermore, the enzyme immunoassay was modifted to measure the specific incorporation of a radioactively labelled precursor ([ 14 C]galactosamine) into chondroitin-6-sulphate. This modification rapidly gives Information on the cellular glycosaminoglycan synthesis in cell culture. Usmg this method our experiments with cultivated human chondrocytes showed that the synthesis of chpndroitin^o-sulphate decreased in the presence of interleukin-la (60.0% less), turnour necrosis factor a (64.4%), -interferön (21.6%) and lipopolysaccharide (53.4%).
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