期刊
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
卷 858, 期 1-2, 页码 32-41出版社
ELSEVIER
DOI: 10.1016/j.jchromb.2007.08.002
关键词
diabetes; enzyme inhibition; michaelis-menten; nitric oxide; s-nitrosothiols; SNACET
Asymmetric dimethylarginine (ADMA), an endoggenous inhibitor of nitric oxide (NO) synthesis, is hydrolyzed to dimethylamine (DMA) and L-Citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). In the present article we report on a GC-MS assay for DDAH activity in rat liver homogenate in phosphate buffered saline. The method is based on the quantitative determination of ADMA-derived DMA by GC-MS as the pentafluorobenzamide derivative. Quantification was performed by selected-ion monitoring of the protonated molecules at ink 240 for DMA and m/z 246 for the internal standard (CD3)(2)NH in the positive-ion chemical ionization mode. The assay was applied to determine the enzyme kinetics in rat liver, the hepatic DDAH activity in streptozotocin-induced (50 mg/kg) diabetes in rats, and to evaluate the importance of S-nitrosothiols as DDAH inhibitors. The K-M and V-max, values were determined to be 60 mu M ADMA and 12.5 pmol DMA/min mg liver corresponding to 166 pmol DMA/min mg protein. Typical DDAH activity values measured in rat liver homogenate were 8.7 pmol DMA/min mg liver at added ADMA concentration of 100 mu M. DDAH activity was found to be 1.7-fold elevated in diabetic as compared to non-diabetic rats (P = 0.01). The SH-specific agents HaCl(2) S-nitrosocysteine ethyl ester (SNACET), a synthetic lipophilic S-nitrosothiol, S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO) and S-nitrosohomocysteine (HcysNO) were found to inhibit DDAH activity in rat liver homogenate. The IC50 values for HcysNO, SNACET, CysNO and GSNO were estimated to be 300, 500, 700 and 1000 mu M, respectively. Oral administration of N-15-labelled SNACET to two healthy volunteers (1 mu mol/kg) resulted in elevated urinary excretion of N-15-labelled nitrite and nitrate, but did not reduce creatinine-corrected excretion of DMA in the urine. Our results suggest that inhibition of DDAH activity on the basis of reversible nitros(yl)ation or irreversible N-thiosulfoximidation of the sulfhydryl group of the cysteine moiety involved in the catalytic process is most likely not a rationale design of DDAH inhibitors. A major advantage of the present GC-MS assay over other assays is that DDAH activity is assessed by measuring the formation of the specific enzymatic product DMA but not the formation of unlabelled or (radio)labelled L-citrulline or the decay of the substrate ADMA. The GC-MS assay reported here should be suitable to probe for DDAH activity in various disease models. (C) 2007 Elsevier B.V. All rights reserved.
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