Bullinger Dino, Fux Richard, Nicholson Graeme, Plontke Stefan, Belka Claus, Laufer Stefan, Gleiter Christoph H, Kammerer Bernd
Department of Clinical Pharmacology, Institute of Pharmacology and Toxicology, University Hospital Tübingen, Tübingen, Germany.
J Am Soc Mass Spectrom. 2008 Oct;19(10):1500-13. doi: 10.1016/j.jasms.2008.06.015. Epub 2008 Jun 28.
The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.
人体对多种疾病的生理反应可通过生物体液中的代谢物模式反映出来。癌症与其他伴有代谢紊乱的疾病一样，会对细胞更新率、修饰酶活性以及RNA/DNA修饰产生特征性影响。这导致修饰核苷和生化相关化合物的排泄发生改变。在我们的代谢谱分析项目中，我们对肺癌、直肠癌或头颈癌患者的24小时尿液进行筛查，以寻找此前未知的核糖基化代谢物。因此，我们开发了一种基于硼酸酯亲和色谱的样品制备方法，随后用制备型薄层色谱进行额外的预纯化。通过离子阱质谱（IT MS）和傅里叶变换离子回旋共振质谱（FTICR MS）对分离出的代谢物进行分析。IT MS用于液相色谱-自动串联质谱（LC-auto MS(3)）筛查运行和质谱（MS(n(n = 4 - 6))）注射泵进样实验，产生特征性碎片模式。FTICR MS测量能够基于精确质量测定计算相应的分子式（质量准确度：外部为1 - 5 ppm，内部校准为亚ppm值）。我们能够鉴定出22种源自细胞RNA代谢以及相关代谢途径（如组氨酸代谢、嘌呤生物合成、甲硫氨酸/多胺循环和烟酸/烟酰胺代谢）的代谢物。发现化合物1 - 核糖基 - 3 - 羟基吡啶鎓、1 - 核糖基 - 吡啶鎓和3 - 核糖基 - 1 - 甲基 - l - 组氨酸鎓以及一系列在N(ω)位与羧酸共轭的核糖基化组胺为新型尿液成分。首次证实了修饰核苷2 - 甲硫基 - N(6) - (顺式 - 羟基异戊烯基) - 腺苷、5 - 甲氧基羰基甲基 - 2 - 硫代尿苷、N(6) - 甲基 - N(6) - 苏氨甲酰腺苷和2 - 甲硫基 - N(6) - 苏氨甲酰腺苷在人尿液中的存在。
