Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 851, Issue 1-2, Pages 220-228Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2007.01.020
Keywords
L-arginine; ADMA; SDMA; assay; stable isotopes; mass spectrometry
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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.
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