Centre de Recherche en Nutrition Humaine Rhône-Alpes, F-69310 Pierre Bénite, France.
Rapid Commun Mass Spectrom. 2009 Dec;23(23):3855-67. doi: 10.1002/rcm.4294.
A reference method to accurately define kinetics in response to the ingestion of glucose in terms of total, exogenous and endogenous glucose is to use stable-isotope-labelled compounds such as 2H and 13C glucose followed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. The use of the usual pentaacetyl (5Ac) derivative generates difficulties in obtaining accurate and reproducible results due to the two chromatographic peaks for the syn and anti isomers, and to the isotopic effect occurring during acetylation. Therefore, the pentaacetylaldononitrile derivative (Aldo) was validated for both isotopes, and compared with the 5Ac derivative. A correction factor including carbon atom dilution (stoichiometric equation) and the kinetic isotopic effect (KIE) was determined. Analytical validation results for the 2H GC/MS and 13C GC/C/IRMS measurements produced acceptable results with both derivatives. When 2H enrichments of plasma samples were < or = 1 mol % excess (MPE), the repeatability (RSD(Aldo Intra assay and Intra day) <0.94%, RSD(5Ac Intra assay and Intra day) <3.29%), accuracy (Aldo <3.4%, 5Ac <29.0%), and stability of the derivatized samples were significantly better when the Aldo derivatives of the plasma samples were used (p < 0.05). When the glucose kinetics were assessed in nine human subjects, after glucose ingestion, the plasma glucose 2H enrichments were identical with both derivatives, whereas the 13C enrichments needed a correction factor to fit together. Due to KIE variation, this correction factor was not constant and had to be calculated for each batch of analyses, to obtain satisfactory results. Mean quantities of exogenous glucose exhibit marked difference (20.9 +/- 1.3g (5Ac) vs. 26.7 +/- 2.5g (Aldo)) when calculated with stoichiometric correction, but fit perfectly when calculated after application of the correction factor (22.1 +/- 1.3g (5Ac) vs. 22.9 +/- 1.9g (Aldo)). Finally, the pentaacetylaldononitrile derivative, used here in GC/C/IRMS for the first time, enables measurement of 2H and 13C enrichments in plasma glucose with a single sample preparation.
一种参考方法是使用稳定同位素标记的化合物（如 2H 和 13C 葡萄糖），然后通过气相色谱/质谱（GC/MS）和气相色谱/燃烧/同位素比质谱（GC/C/IRMS）分析，准确地定义葡萄糖摄入时的总葡萄糖、外源性葡萄糖和内源性葡萄糖动力学。由于顺式和反式异构体的两个色谱峰以及乙酰化过程中的同位素效应，使用常用的五乙酰基（5Ac）衍生物会导致难以获得准确和可重复的结果。因此，验证了戊乙酰基醛腈衍生物（Aldo）对于这两种同位素，并与 5Ac 衍生物进行了比较。确定了包括碳原子稀释（化学计量方程式）和动力学同位素效应（KIE）在内的校正因子。两种衍生物的 2H GC/MS 和 13C GC/C/IRMS 测量的分析验证结果均令人满意。当血浆样品的 2H 丰度 <或= 1 mol%过量（MPE）时，当使用 Aldo 衍生物时，血浆样品衍生化的重复性（Aldo 日内和日内测定的 RSD<0.94%，5Ac 日内和日内测定的 RSD<3.29%）、准确性（Aldo<3.4%，5Ac<29.0%）和稳定性均显著更好（p<0.05）。当在九名人类受试者中评估葡萄糖动力学时，葡萄糖摄入后，两种衍生物的血浆葡萄糖 2H 丰度相同，而 13C 丰度需要校正因子才能拟合在一起。由于 KIE 变化，该校正因子不是常数，必须为每批分析计算，以获得满意的结果。用化学计量校正计算时，外源性葡萄糖的平均量差异明显（5Ac 为 20.9 +/- 1.3g，Aldo 为 26.7 +/- 2.5g），但应用校正因子后完全吻合（5Ac 为 22.1 +/- 1.3g，Aldo 为 22.9 +/- 1.9g）。最后，戊乙酰基醛腈衍生物，首次在此用于 GC/C/IRMS，可通过单个样品制备测量血浆葡萄糖中的 2H 和 13C 丰度。
