Maas Renke, Tan-Andreesen Jing, Schwedhelm Edzard, Schulze Friedrich, Böger Rainer H
Institute of Experimental and Clinical Pharmacology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 May 15;851(1-2):220-8. doi: 10.1016/j.jchromb.2007.01.020. Epub 2007 Jan 26.
The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for the hydrolysis of asymmetric dimethylarginine (ADMA) to L-citrulline and dimethylamine. DDAH is currently investigated as a promising target for therapeutic interventions, as ADMA has been found to be elevated in cardiovascular disease. In many tissues continuous endogenous formation of ADMA and L-citrulline poses considerable limitations to the presently used assays for DDAH activity, which are commonly based on the measurement of ADMA or L-citrulline. We therefore developed a stable-isotope-based assay suitable for 96-well plates to determine DDAH activity. Using deuterium-labeled ADMA ([(2)H(6)]-ADMA) as substrate and double stable-isotope labeled ADMA ([(13)C(5)-(2)H(6)]-ADMA) as internal standard we were able to simultaneously determine formation and metabolism of ADMA in renal and liver tissue of mice by LC-tandem MS. Endogenous formation of ADMA could largely be abolished by addition of protease inhibitors, while metabolism of [(2)H(6)]-ADMA was not significantly altered. The intra-assay coefficient of variation for the determination of endogenous ADMA and [(2)H(6)]-ADMA was 2.4% and 4.8% in renal and liver tissue, respectively. The inter-assay coefficient of variation for DDAH activity based on degradation of [(2)H(6)]-ADMA determined in separate samples from the same organs was determined to be 8.9% and 10% for mouse kidney and liver, respectively. The present DDAH activity assay allows for the first time to simultaneously determine DDAH activity and endogenous formation of ADMA, SDMA, and L-arginine in tissue.
二甲基精氨酸二甲胺水解酶（DDAH）负责将不对称二甲基精氨酸（ADMA）水解为L-瓜氨酸和二甲胺。目前，DDAH作为一种有前景的治疗干预靶点正在被研究，因为已发现ADMA在心血管疾病中水平升高。在许多组织中，ADMA和L-瓜氨酸的持续内源性生成对目前常用的DDAH活性检测方法造成了很大限制，这些方法通常基于对ADMA或L-瓜氨酸的测量。因此，我们开发了一种适用于96孔板的基于稳定同位素的检测方法来测定DDAH活性。使用氘标记的ADMA（[(2)H(6)]-ADMA）作为底物，双稳定同位素标记的ADMA（[(13)C(5)-(2)H(6)]-ADMA）作为内标，我们能够通过液相色谱-串联质谱同时测定小鼠肾和肝组织中ADMA的生成和代谢情况。添加蛋白酶抑制剂后，ADMA的内源性生成在很大程度上可以被消除，而[(2)H(6)]-ADMA的代谢没有显著改变。在肾和肝组织中，测定内源性ADMA和[(2)H(6)]-ADMA的批内变异系数分别为2.4%和4.8%。基于在来自同一器官的单独样本中[(2)H(6)]-ADMA降解情况测定的DDAH活性的批间变异系数，小鼠肾和肝分别为8.9%和10%。目前的DDAH活性检测方法首次能够同时测定组织中DDAH活性以及ADMA、SDMA和L-精氨酸的内源性生成情况。
