This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: A 1-mL solution of the NANA sample with a concentration of 0.03 ?g/?L was prepared and was used for analysis of N-acetylneuraminic acid content. The NANA sample was not subjected to any hydrolysis step. For the SALIVA sample, two 200-?g aliquots were prepared for neutral and amino sugars, and sialic acids analyses. The saliva aliquot for neutral and amino sugars analysis was hydrolyzed with 2N trifluoroacetic acid at 100oC for 4 hr, whereas the aliquot for sialic acids analysis was hydrolyzed with 2M acetic acid at 80oC for 3 hr. All hydrolysates were dried under nitrogen gas thereafter, resuspended in H2O, sonicated for 5 min in ice and transferred to injection vials. A mix of unhydrolyzed standards for N-acetylneuraminic acid and N-glycolylneuraminic acid was run at the same time as the NANA sample. Another mix of standards for neutral and amino sugars, and N-acetylneuraminic acid and N-glycolylneuraminic acid were hydrolyzed in the same manner and at the same time as the SALIVA sample. Four concentrations of the standard mixtures were prepared to establish calibration equations. The number of moles of each analyte in the NANA or SALIVA sample was quantified by linear interpolation from the calibration equation. The neutral and amino sugars, and sialic acids were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The monosaccharide residues were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used the following mobile phase eluents: A, degassed nanopure water and B, 200 mM NaOH for neutral and amino sugars;and C, 100 mM NaOH, and D, 1M sodium acetate in 100 mM NaOH for sialic acids analysis. Injections were made every 45 min for neutral and amino sugars and every 40 min for sialic acids analysis. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., """"""""High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates"""""""", 1994, Methods Enzymol. 230: 208-225).
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