A stable-isotope based technique for the determination of dimethylarginine dimethylaminohydrolase (DDAH) activity in mouse tissue
A stable-isotope based technique for the determination of dimethylarginine dimethylaminohydrolase (DDAH) activity in mouse tissue
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DOI：
10.1016/j.jchromb.2007.01.020
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发表时间：
2007-05-15
影响因子：
3
通讯作者：
Boeger, Rainer H.
中科院分区：
文献类型：
--
作者：
Maas, Renke;Tan-Andreesen, Jing;Boeger, Rainer H.
二甲基精氨酸二甲基氨基水解酶（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。DDAH目前被研究为治疗干预的有希望的靶点，因为已经发现ADMA在心血管疾病中升高。在许多组织中，ADMA和L-瓜氨酸的连续内源性形成对目前使用的DDAH活性测定造成了相当大的限制，DDAH活性测定通常基于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测定法来测定DDAH活性。以氘标记的ADMA（[H-2（6）]-ADMA）为底物，双稳定同位素标记的ADMA（[C-13（5）-H-2（6）]-ADMA）为内标物，采用液相色谱-串联质谱法同时测定了小鼠肾和肝组织中ADMA的形成和代谢。而[H-2（6）]-ADMA代谢无明显改变。在肾脏和肝脏组织中，测定内源性ADMA和[H-2（6）]-ADMA的批内变异系数分别为2.4%和4.8%。对于小鼠肾脏和肝脏，基于在来自相同器官的单独样品中测定的[H-2（6）]-ADMA降解的DDAH活性的试验间变异系数分别为8.9%和10%。本DDAH活性测定首次允许同时测定DDAH活性和组织中ADMA、SDMA和L-精氨酸的内源性形成。(C)2007 Elsevier B. V.保留所有权利。
The enzyme dimethylarginine dimethylarnmohydrolase (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 ([H-2(6)]-ADMA) as substrate and double stable-isotope labeled ADMA ([C-13(5)-H-2(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 [H-2(6)]-ADMA was not significantly altered. The intra-assay coefficient of variation for the determination of endogenous ADMA and [H-2(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 [H-2(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. (C) 2007 Elsevier B.V. All rights reserved.