Identification of N-acetylhexosamines produced by enzymes of the N-acetylneuraminic acid metabolic pathway by borate complex anion-exchange chromatography of the corresponding N-acetylhexosaminitols.
A mixture of hexosaminitols obtained by reducing N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine with sodium borohydride was resolved by borate complex anion-exchange chromatography. This procedure yielded a complete separation of N-acetylglucosaminitol, N-acetylgalactosaminitol, and N-acetylmannosaminitol and provided a rapid and accurate means for identifying and measuring N-acetylhexosamines in biological samples. This method was applied to studies on N-acetylneuraminic acid metabolism in human skin fibroblasts. It was used to identify reaction products in two enzymatic reactions: the conversion of UDP-N-acetylglucosamine to N-acetylmannosamine and UDP by UDP-N-acetylglucosamine 2-epimerase and the conversion of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate by N-acetylneuraminate pyruvate-lyase. It was also used to identify the free 3H-labeled N-acetylhexosamines found in fibroblasts cultured in the presence of N-[3H]acetylmannosamine.
D001881:Borates; D002238:Carbohydrate Epimerases / Q000378:metabolism; D002352:Carrier Proteins; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D006595:Hexosamines / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D019158:N-Acetylneuraminic Acid; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D012867:Skin / Q000201:enzymology / Q000378:metabolism; D013402:Sugar Alcohols / Q000032:analysis*
D001881:Borates; D002352:Carrier Proteins; D006595:Hexosamines; D012794:Sialic Acids; D013402:Sugar Alcohols; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; C498259:RENBP protein, human; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine