Kammerer Bernd, Frickenschmidt Antje, Müller Christa E, Laufer Stefan, Gleiter Christoph H, Liebich Hartmut
Department of Clinical Pharmacology, University Hospital Tübingen, Otfried-Müller-Str. 45, 72076 Tübingen, Germany.
Anal Bioanal Chem. 2005 Jun;382(4):1017-26. doi: 10.1007/s00216-005-3232-2. Epub 2005 May 19.
In diseases accompanied by strong metabolic disorders, like cancer and AIDS, modifying enzymes are up- or down-regulated. As a result, many different types of metabolic end-products, including abnormal amounts of modified nucleosides, are found in urine. These nucleosides are degradation products of an impaired ribonucleic acid (RNA) metabolism, which affects the nucleoside pattern in urine. In several basic experiments we elucidated the fragmentation pathways of 16 characteristic nucleosides and six corresponding nucleic bases that occur in urine using electrospray ionization ion trap MS(5) (ESI-ITMS) experiments operated in positive ionization mode. For urinary nucleoside analysis, we developed an auto-LC-MS3 method based on prepurification via boronate gel affinity chromatography followed by reversed phase chromatography. For this purpose, an endcapped LiChroCART Superspher RP 18 column with a gradient of ammonium formate and a methanol-water mixture was used. This method gives a limit of detection of between 0.1 and 9.6 pmol for 15 standard nucleosides, depending on the basicity of the nucleoside. Overall, the detection of 36 nucleosides from urine was feasible. It was shown that this auto-LC-MS3 method is a valuable tool for assigning nucleosides from complex biological matrices, and it may be utilized in the diagnosis of diseases associated with disorders in RNA metabolism.
在伴有强烈代谢紊乱的疾病中，如癌症和艾滋病，修饰酶会被上调或下调。结果，在尿液中发现了许多不同类型的代谢终产物，包括异常数量的修饰核苷。这些核苷是核糖核酸（RNA）代谢受损的降解产物，会影响尿液中的核苷模式。在一些基础实验中，我们使用正离子模式运行的电喷雾电离离子阱质谱（ESI-ITMS）实验，阐明了尿液中出现的16种特征核苷和6种相应核酸碱基的裂解途径。对于尿液核苷分析，我们开发了一种自动液相色谱-串联质谱（LC-MS3）方法，该方法基于通过硼酸酯凝胶亲和色谱进行预纯化，然后进行反相色谱分析。为此，使用了带有甲酸铵和甲醇-水混合物梯度的封端LiChroCART Superspher RP 18柱。该方法对15种标准核苷的检测限在0.1至9.6皮摩尔之间，具体取决于核苷的碱性。总体而言，从尿液中检测36种核苷是可行的。结果表明，这种自动LC-MS3方法是从复杂生物基质中鉴定核苷的有价值工具，可用于诊断与RNA代谢紊乱相关的疾病。
