Ceglarek Uta, Dittrich Julia, Helmschrodt Christin, Wagner Kristin, Nofer Jerzy-Roch, Thiery Joachim, Becker Susen
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany.
Center of Laboratory Medicine, University Hospital Münster, Albert-Schweizer-Campus 1, 48129 Münster, Germany.
Clin Chim Acta. 2014 Aug 5;435:1-6. doi: 10.1016/j.cca.2014.04.010. Epub 2014 Apr 24.
Preanalytical standardization is required for a reliable quantification of the signaling molecules sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and sphingosine (SPH).
Methanolic protein precipitation of 15μL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3min by hydrophilic interaction liquid chromatography (HILIC, SeQuant™ ZIC®-HILIC column) followed by tandem mass spectrometry. Stability of analytes in whole blood and plasma was investigated. Sphingolipid concentrations were determined in human plasma (n=50) and mice deficient in sphingosine kinase 1 (SK1) and 2 (SK2) (n=5).
Storing EDTA whole blood >60min after blood withdrawal at room temperature resulted in an increase in S1P and SPH concentrations of ≥25%. Significant changes in SPH levels of +37% were observed after 60min of storage of EDTA plasma at room temperature. Repeated freeze-thaw cycles of EDTA plasma resulted in increased S1P and SPH levels. Concentrations in human EDTA plasma were between 55.5 and 145.2ng/mL for S1P and between 8.9 and 35.3ng/mL for SA1P. Concentrations of S1P were 36% lower and 96% higher in EDTA-plasma from SK1- and SK2-deficient mice, respectively, compared to the wild type.
Preanalytical standardization is a precondition for the analysis of sphingolipids in human blood.
为了可靠地定量测定信号分子鞘氨醇-1-磷酸（S1P）、鞘氨醇胺-1-磷酸（SA1P）和鞘氨醇（SPH），需要进行分析前标准化。
在分析前，采用甲醇沉淀法对15μL乙二胺四乙酸（EDTA）血浆中的蛋白质进行沉淀。通过亲水相互作用液相色谱法（HILIC，SeQuant™ ZIC®-HILIC柱）在3分钟内分离鞘脂，随后进行串联质谱分析。研究了分析物在全血和血浆中的稳定性。测定了人血浆（n = 50）以及鞘氨醇激酶1（SK1）和2（SK2）缺陷小鼠（n = 5）中的鞘脂浓度。
采血后在室温下储存EDTA全血超过60分钟，导致S1P和SPH浓度增加≥25%。在室温下储存EDTA血浆60分钟后，观察到SPH水平有37%的显著变化。EDTA血浆反复冻融循环导致S1P和SPH水平升高。人EDTA血浆中S1P的浓度在55.5至145.2 ng/mL之间，SA1P的浓度在8.9至35.3 ng/mL之间。与野生型相比，SK1缺陷小鼠和SK2缺陷小鼠的EDTA血浆中S1P的浓度分别降低了36%和升高了96%。
分析前标准化是分析人血中鞘脂的前提条件。
