DETERMINATION OF D-MANNITOL, 3 METHYL GLUCOSE AND LACTULOSE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 富含D-甘露醇、3-甲基葡萄糖和乳果糖的原始猴血浆的富集 用不同浓度的D-甘露醇、3-甲基葡萄糖和乳果糖富集3份500-<$L原始猴血浆。 计算加入的糖在血浆浓度1中的浓度如下：D-甘露醇[0.012 <$g/<$L]、3-甲基葡萄糖[0.035 <$g/<$L]和乳果糖[0.014 <$g/<$L];浓度2：D-甘露醇[0.024 <$g/<$L]、3-甲基葡萄糖[0.070 <$g/<$L]和乳果糖[0.028 <$g/<$L]; Conc 3：D-甘露醇[0.048 <$g/<$L]、3-甲基葡萄糖[0.140 <$g/<$L]和乳果糖[0.056 <$g/<$L]。 血浆的脱蛋白和脱盐 向富集的血浆中加入等体积的冰冷的5-磺基水杨酸（35 g/L），并使混合物在冰上凝固10 min。随后将冷的混合物在2800 rpm、4 ℃下旋转30 min。 立即将上清液与约4 mL IONAC（预先用OH-活化）混合以除去阴离子，并将各浓度的所有液体部分转移至另一管中并冻干。 通过高pH阴离子交换色谱-脉冲安培检测（HPAEC-PAD）分析单糖组成 用500 μ L纳米纯水溶解各浓度的干燥富集血浆（以恢复原始血浆体积），并通过HPAEC-PAD分析D-甘露醇、3-甲基葡萄糖和乳果糖。 在水中制备糖标准品混合物，最高浓度（甘露醇甲基葡萄糖乳果糖标准品4）如下：D-甘露醇= 0.50 <$g/10 <$L，3-甲基葡萄糖= 1.00 <$g/10 <$L，乳果糖= 0.75 <$g/10 <$L。 通过连续稀释制备总共四种标准混合物，每次进一步稀释为先前较高浓度的50%。 通过内插到校准方程中，定量测定血浆中各残留物的浓度。 先前单独分析每种糖标准品以确定其保留时间。 通过使用配备有梯度泵、电化学检测器和自动进样器的Dionex ICS 3000系统分析糖。 通过具有氨基捕集器的Dionex CarboPac PA 20（3 X 150 mm）分析柱分离各个糖。 用10 mM NaOH梯度通过色谱柱10 min，18 min后升至100 mM。用400 mM NaOAc清洗色谱柱8 min，最终再平衡至10 mM，总运行周期为50 min。所有标准品混合物和浓度处理的进样体积为10 µ L。

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.