Low Sulfated Glycosaminoglycans Research Articles

Heparins, low-molecular-weight heparins, and other glycosaminoglycans analyzed by agarose gel electrophoresis and azure A-silver staining.

A sensitive, nonradioactive azure A-silver staining method combining agarose gel electrophoresis was established and evaluated. Unfractionated heparins (UFHs), low-molecular-weight heparins (LMWHs), heparan sulfate (HS), chondroitin sulfate A (CSA), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronic acid (HA) were analyzed. The detection limit of the method was 0.5 ng for heparin, LMWH, HA, CSA, and DS, 2 ng for KS, and 6 ng for HA in the 2-microliter sample volume. Dilution curves demonstrated linear correlation between the logarithm of the concentration of glycosaminoglycans (GAGs) and their optical absorbance at 548 nm. The linear ranges were 1 to 500 ng/microliter for heparins, LMWHs, HS, DS, and CSA, 3 to 500 ng/microliter for KS, and 8 to 500 ng/microliter for HA. GAGs have their characteristic migration patterns and their Rf value decreased from CSA to KS, DS, HS, heparin, and HA. The differences were described for heparins and LMWHs. LMWHs migrated faster and displayed broader bands than unfractionated heparins. It was also observed that some unfractionated heparins contained low sulfated GAGs as contamination, which seemed to be DS as judged by their migration patterns.

Synthesis of aberrant glycosaminoglycans during cartilage culture in 'sulfate free' medium.

Incorporation of radiolabeled sulfate into glycosaminoglycans is a widely accepted assay to measure the rate of proteoglycan synthesis. Although glycosaminoglycan synthesis is dependent on the quantity of inorganic sulfate available to proteoglycan synthesizing cells, 'sulfate free' medium is regularly used in studies regarding proteoglycan synthesis. In this study murine patellar cartilage glycosaminoglycans synthesized under 'sulfate free' conditions were compared with those synthesized at physiological sulfate concentration. Under 'sulfate free' conditions synthesis was not only decreased but low sulfated glycosaminoglycans were made that were not synthesized during incubation at physiological sulfate concentration. The use of 'sulfate free' medium should be avoided in proteoglycan synthesis studies.

Low sulfated glycosaminoglycans are excreted in patients with the Lowe syndrome

Glycosaminoglycans (GAGs) were prepared from the urine of three patients and from normal individuals by cetylpyridinium chloride precipitation and Pronase digestion. The GAGs were analyzed by electrophoresis, anion-exchange chromatography, and enzymatic and chemical degradation. Each of the three patients showed a four- to fivefold increase in urinary GAG excretion compared to normal controls and in one patient a tenfold increase was measured during a period of behavioral agitation which included joint swelling. Urinary GAGs from affected individuals were characterized by a high proportion of low sulfated molecules. The predominant low sulfated component was chondroitin-4-sulfate (C4S); however, small amounts of chondroitin-6-sulfate (C6S) were also present. Heparan sulfate (HS) was present in normal proportion (5–10%) and most of it was not low sulfated. Abnormal excretion of chondroitin (Ch), hyaluronic acid (HA), and dermatan sulfate (DS) was not detected. These findings suggest that the clinical manifestations of Lowe syndrome may be caused by a defect in GAG metabolism.