Determination of NG,NG-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column.
A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.
Nonaka
Satoko
S
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
Tsunoda
Makoto
M
Aoyama
Chiaki
C
Funatsu
Takashi
T
D000581:Amidohydrolases / Q000032:analysis*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007668:Kidney / Q000201:enzymology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012822:Silicon Dioxide
C018524:N,N-dimethylarginine; D012822:Silicon Dioxide; D001120:Arginine; D000581:Amidohydrolases; C059999:dimethylargininase