Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All.
A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.
Volpi
Nicola
N
Department of Biologia Animale, University of Modena and Reggio Emilia, Via Campi 213/d, 41100 Modena, Italy. volpi@unimo.it
Maccari
Francesca
F
Titze
Jens
J
D000818:Animals; D002232:Carbocyanines; D004396:Coloring Agents; D003871:Dermatan Sulfate / Q000032:analysis*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006025:Glycosaminoglycans / Q000032:analysis; D006820:Hyaluronic Acid / Q000032:analysis*; D008297:Male; D008832:Microchemistry / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods; D014048:Tolonium Chloride
D002232:Carbocyanines; D004396:Coloring Agents; D006025:Glycosaminoglycans; D014048:Tolonium Chloride; D003871:Dermatan Sulfate; C012040:stains-all; D006820:Hyaluronic Acid