A stable-isotope based technique for the determination of dimethylarginine dimethylaminohydrolase (DDAH) activity in mouse tissue.
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.
Maas
Renke
R
Institute of Experimental and Clinical Pharmacology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. maas@uke.uni-hamburg.de
Tan-Andreesen
Jing
J
Schwedhelm
Edzard
E
Schulze
Friedrich
F
Böger
Rainer H
RH
D000581:Amidohydrolases / Q000032:analysis*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D007553:Isotope Labeling / Q000379:methods*; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
C018524:N,N-dimethylarginine; D001120:Arginine; D000581:Amidohydrolases; C059999:dimethylargininase