Schierbeek Henk, Rook Denise, te Braake Frans W J, Dorst Kristien Y, Voortman Gardi, Godin Jean-Philippe, Fay Laurent-Bernard, van Goudoever Johannes B
Erasmus Medical Center - Sophia Children's Hospital, Department of Paediatrics, Division of Neonatology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
Rapid Commun Mass Spectrom. 2009 Sep;23(18):2897-902. doi: 10.1002/rcm.4200.
Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine (13)C-glutathione as its dimeric form (GSSG) and its precursor [1-(13)C]glycine in a small volume of erythrocytes in one single analysis. After having transformed (13)C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the (13)C-isotopic enrichment of GSSG and glycine in 150 microL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of micromol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 micromol/mL. The (13)C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3 per thousand) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants.
使用稳定同位素测定谷胱甘肽动力学需要准确测量示踪剂和被追踪物。以前，前体和合成产物是用两种不同的技术进行测量的，即液相色谱/同位素比率质谱法（LC/IRMS）和气相色谱/燃烧/同位素比率质谱法（GC/C/IRMS）。为了减少样品体积并将分析工作量降至最低，我们开发了一种方法，可在一次单一分析中同时测定少量红细胞中作为其二聚体形式（GSSG）的（13）C-谷胱甘肽及其前体[1-（13）C]甘氨酸。在将（13）C-谷胱甘肽转化为其二聚体形式GSSG后，我们使用与LC/IRMS联用的液相色谱法测定了150微升全血中GSSG和甘氨酸的细胞内浓度以及（13）C-同位素丰度。结果表明，使用环亮氨酸作为内标可可靠地测量浓度（微摩尔/毫升范围），即精密度优于0.1微摩尔/毫升。在同一次运行中测量的GSSG和甘氨酸的（13）C-同位素丰度给出了具有出色精密度（标准偏差（sd）<0.3‰）和准确度（测量值在零至5 APE之间）的可靠值。这种新方法通过相对低剂量施用示踪剂开辟了各种动力学研究，降低了研究设计的总成本。此外，仅需要最小的样品体积，甚至能够对非常小的受试者，如早产儿进行研究。
