In vitro determination of asymmetric dimethylarginine (ADMA) breakdown by dimethylarginine dimethylaminohydrolase (DDAH) activity in rat kidney; evidence for inhibition by superoxide anion (O∗) and nitric oxide (NO)

Background: ADMA is an endogenous inhibitor of NO synthase (NOS) that is importantly metabolized by DDAH in kidney. ADMA breakdown yields L‐citrulline (CIT) and rate of CIT production in kidney homogenates has been used as a measure of DDAH activity. Materials and Methods: A colorimetric method used to detect CIT relies on diacetyl monoxime (DAMO) but color also develops with urea; an obvious problem for kidney measurements. We investigated use of urease (to degrade urea) and also whether O∗ and NO could inhibit DDAH activity, as previously shown in endothelial cell culture. Results: Urea causes a high background color with DAMO and incubation with urease (100U/ml homogenate containing 2mg protein/ml) reduces the background to zero. We have also determined the ideal method of deproteinization (4% sulfosalicylic acid), incubation time (45min) and homogenization buffer (Na2PO4). Using our optimized conditions there is a strong correlation between rate of CIT accumulation and rate of ADMA breakdown (p=0.003; slopes are 0.395 and 0.398 μmol/g protein/min). The NO donors NONOATE and NaNO2 produce dose dependent inhibition of DDAH activity and O∗ (from the O∗ generator DMNQ) also inhibits DDAH activity. Conclusions: When urease is used, rate of CIT formation using DAMO gives an accurate measure of DDAH activity in kidney homogenate and both oxidation and nitrosylation acutely (within 45min) inhibit DDAH activity.