DETERMINATION OF D-MANNITOL, 3 METHYL GLUCOSE AND LACTULOSE

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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: Enrichment of Raw Monkey Plasma enriched with D-mannitol, 3-methyl glucose, and lactulose Three 500-¿L raw monkey plasmas were enriched with different concentrations of D-mannitol, 3-methyl glucose, and lactulose. The sugars added were calculated to have the following concentrations in the plasma ¿ Conc 1: D-mannitol [0.012 ¿g/¿L], 3-methyl glucose [0.035 ¿g/¿L], and lactulose [0.014 ¿g/¿L]; Conc 2: D-mannitol [0.024 ¿g/¿L], 3-methyl glucose [0.070 ¿g/¿L], and lactulose [0.028 ¿g/¿L]; and Conc 3: D-mannitol [0.048 ¿g/¿L], 3-methyl glucose [0.140 ¿g/¿L], and lactulose [0.056 ¿g/¿L]. Deproteinization and Desalting of Plasma The enriched plasmas were added with equal volumes of ice-cold 5-sulfosalicylic acid (35g/L) and the mixtures were allowed to set in ice for 10 min. The cold mixtures were spun subsequently at 2800 rpm, 4oC, for 30 min. The supernatants were immediately mixed with about 4 mL of IONAC (previously activated with OH-) to remove anions and all the liquid portion for each concentration was transferred into another tube and lyophilized. Monosaccharide Composition Analysis by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) The dried enriched plasma for each concentration was dissolved with 500 ¿L nanopure water (to restore the original plasma volume) and analyzed for D-mannitol, 3-methyl glucose, and lactulose by HPAEC-PAD. A mixture of sugar standards was prepared in water with the highest concentrations (MannitolMethGlcLactuloseStd4) being the following: D-mannitol=0.50 ¿g/10 ¿L, 3-methyl glucose=1.00 ¿g/10 ¿L, and lactulose=0.75 ¿g/10 ¿L. A total of four standard mixtures were prepared by serial dilution with each further dilution being 50% of the previous higher concentration. The concentration of each residue in the plasma was quantified by interpolation into the calibration equation. Each sugar standard was previously analyzed individually to determine their retention times. The sugars were analyzed by using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual sugars were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The column was passed with a gradient of 10 mM NaOH for 10 min, which was ramped to 100 mM by 18 min. The column was cleaned with 400 mM NaOAc for 8 min and eventually re-equilibrated to 10 mM for a total run cycle of 50 min. Injection volume for all standard mixtures and concentration-treatments was 10 ¿L.