Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography–mass spectrometry

Serum L-Homoarginine Concentration is Elevated During Normal Pregnancy and is Related to Flow-Mediated Vasodilatation

“…The concentrations of ADMA were similar in all trimesters, whereas slight, probably clinically unimportant, biphasic changes were observed in maternal SDMA concen- trations, with the second trimester level being statistically significantly lower than those for the third trimester or in the non-pregnant state. The L-arginine/ADMA ratio, an indicator of NO production by NOS, 31 remained unchanged in all trimesters. The ADMA/SDMA ratio, an index of the ADMA degrading enzyme, dimethylarginine dimethylaminohydrolase activity, 32 displayed a statistically significant increase in the second trimester compared with the ratio in the first and third trimesters (p<0.05).…”

Does the Inhibitory Action of Asymmetric Dimethylarginine (ADMA) on the Endothelial Nitric Oxide Synthase Activity Explain Its Importance in the Cardiovascular System? The ADMA Paradox

“…By means of a reliable GC-MS assay (19), we performed detailed mechanistic studies on the inhibitory action of ADMA on a commercially available recombinant bovine eNOS. The results of these studies are shown in Figures 1-3 /min × mg eNOS) is about 300 times smaller than that of a recombinant rat nNOS (19), indicating that eNOS is much less active compared to nNOS. ADMA and L-NMA were found to be equipotent inhibitors of eNOS activity (Figure 1).…”

“…The IC 50 value for ADMA in rat brain homogenates has been reported to be 2 μM (22). Our group found that ADMA is a potent non-competitive inhibitor (K i 0.4 μM; K ii 1.6 μM; IC 50 1.5 μM) of recombinant rat nNOS (3,19,23,24). By contrast, others have reported that ADMA is a weak competitive inhibitor (K i 0.7 μM; IC 50 ≈ 20 μM) of rat nNOS (25).…”

“…eNOS assays were performed with minor modifications of the GC-MS method recently developed for and applied to nNOS (19). Fresh solutions of NOS cofactors were prepared daily in potassium phosphate buffer (50 mM, pH 7, 5 mL), resulting in the following final concentrations: H 4 B, 10 μM; FAD, 5 μM; FMN, 5 μM; CaCl 2 , 500 μM; NADPH, 0.8 mM; calmodulin, 500 nM.…”

“…This reaction is considered particularly important because it diminishes NO bioavailability. Besides ADMA, there are additional endogenous and exogenous N G -substituted L-arginine inhibitors of NOS activity (1,(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)). Yet, only ADMA has been suggested as a potent risk factor for the development of NOassociated endothelial dysfunction and cardiovascular diseases in humans due to its inhibitory action on eNOS (20,21).…”