Determination of the enrichment of the hydrogen bound to carbon 5 of glucose on 2H2O administration.
Determination of the enrichment of the hydrogen bound to carbon 5 of glucose on 2H2O administration.
复制标题
测定 2H2O 给药时与葡萄糖碳 5 结合的氢的富集度。
DOI：
10.1006/abio.2001.5326
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Landau,BR
中科院分区：
文献类型：
--
作者：
Schumann,WC;Gastaldelli,A;Chandramouli,V;Previs,SF;Pettiti,M;Ferrannini,E;Landau,BR
We (1, 2) introduced a method for quantitating the contribution of gluconeogenesis to glucose production. Its use is being increasingly reported (eg, 3–7). The method requires determining the enrichment of the hydrogen bound to carbon 5 of blood glucose following administration of 2H2O. To accomplish this the glucose is converted to 1, 2: 5, 6-di-O-isopropylideneglucofuranose (Fig. 1). The terminal isopropylidene group is then hydrolyzed to yield 1, 2-O-isopropylideneglucofuranose. Oxidation with periodate cleaves the exposed glycol to yield 1, 2-O-isopropylidene-5-aldoxylofuranose which upon reduction with sodium borohydride yields 1, 2-O-isopropylidenexylofuranose. Hydrolysis yields xylose, which on periodate oxidation yields formaldehyde containing carbon 5 of the glucose with its hydrogen. The formaldehyde is condensed with NH3 to form hexamethylenetetramine (HMT). 2 The HMT is assayed for its enrichment by mass spectrometry. Recently, Xie et al.(8) reported a procedure based upon selective hydrolysis by periodic acid in ethyl acetate of the terminal O-isopropylidene group of acyclic carbohydrates containing internal acetals. They reported, working at a millimole scale, with the addition of a reduction step, the dehomologation of 1, 2: 5, 6-di-O-isopropylideneglucofuranose to 1, 2-O-isopropylidenexylofuranose, without purification of intermediates, and in a yield 95% of theoretical. We have adapted this procedure to a micromole scale. The method for determining the enrichment of the hydrogen bound to carbon 5 of glucose then becomes simpler and much less time consuming. Methods. Glucose (0.5–2.0 mg) is stirred for 4 h in 1 ml of acetone to which 40 μl of concentrated H2SO4 has been added in the cold. Then 5 ml of acetone is added.After the solution is cooled, 10 N NaOH is added dropwise with stirring until the deep yellow color of the solution fades. About 0.3 ml of the NaOH is required. The acetone layer (upper layer) is pipetted from the Na2SO4 slurry (lower layer). The slurry is washed with 2 ml of acetone. The pooled acetone layer and wash are evaporated to dryness. The residue is dissolved in 2 ml of ethyl acetate and anhydrous Na2SO4 is added to dry the solution. The solution, which contains the 1, 2: 5, 6-di-O-isopropylideneglucofuranose, is transferred to a vial along with a wash with 1 ml of ethyl acetate of the Na2SO4 pellet. Thirty milligrams of periodic acid is stirred in 10 ml of ethyl acetate until it is dissolved. About 2 h of stirring is required for complete soution. The solution is stable at 20 C for several weeks. It should be discarded when it turns yellow. The periodic acid solution, 0.5 ml and therefore containing 1.5 mg of periodic acid, is added per milligram of starting glucose to the ethyl acetate solution of the 1, 2: 5, 6-di-O-isopropylideneglucofuranose. The mixture is stirred at room temperature for 2 h. An initial bright yellow color gradually fades as a white precipitate forms. The precipitate is removed by filtration through glass wool. The residue, on evaporation, is dissolved in 1 ml of absolute ethanol, 2 mg of NaBH4 in 0.5 ml of absolute ethanol is added, and the mixture is stirred for 30 min at room temperature. Excess NaBH4 is destroyed by dropwise addition of 3 N acetic acid until effervescence ceases. About 0.35 ml is required. The solution is evaporated and 2 ml of methanol is added twice to the residue and evaporated to remove the methyl borate. The residue, after the evaporation of the methanol, is dissolved in 1 ml of saturated NaCl and extracted twice with 3 ml of ethyl acetate. The ethyl acetate is evaporated. The residue, following addition of 1 ml of 0.1 N H2SO4, is heated for 1 h in a boiling water bath to …
