Table 2. Recommend assay procedures/conditions for the measurement of renal cortical DDAH activity.

Homogenize tissue with 5 × sodium phosphate buffer, pH=6.5 Adjust protein concentration to 20 mg/ml Preincubate urease (100 U/ml homogenate) with tissue homogenate in 37°C water bath for 15 min Add 100 μl sample to 400 μl 1 mm ADMA in sodium phosphate buffer (respective blank is sample omitting ADMA) Incubate mixture in 37°C water bath for 45 min Stop reaction by addition of 0.5 ml of 4% sulfosalicylic acid Vortex and centrifuge at 3000 g for 10 min Add 100 μl supernatant into a 96-well plate in triplicate Serially dilute 100 μm l-citrulline standard to 0, 3.125, 6.25, 12.5, 25, 50, and 100 μm Add 100 μl of standard into the 96-well plate in triplicate Mix one part of oxime reagent with two parts of antipyrine/H2SO4 reagent to make the ‘color mixture’ Add 100 μl of color mixture into the wells Cover the plate with a sealing tape Shake on a plate shaker for 1 min Incubate the plate in 60°C water bath for 110 min in the dark Cool the plate in an ice bath for 10 min Measure the absorbance by spectrophotometric analysis at 466 nm Subtract the value of respective blank The DDAH activity is represented as μm l-citrulline formation/g protein/min at 37°C

ADMA, asymmetric dimethylarginine; DDAH, dimethylarginine dimethylaminohydrolase.