GC–MS assay for hepatic DDAH activity in diabetic and non-diabetic rats by measuring dimethylamine (DMA) formed from asymmetric dimethylarginine (ADMA): Evaluation of the importance of S-nitrosothiols as inhibitors of DDAH activity in vitro and in

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• 作者：Kristine Chobanyan ; Thomas Thum ; Maria-Theresia Suchy ; Bijun Zhu ; Anja Mitschke ; Frank-Mathias Gutzki ; Bibiana Beckmann ; Dirk O. Stichtenoth ; Dimitrios Tsikas
• 关键词：Diabetes ; Enzyme inhibition ; Michaelis– ; Menten ; Nitric oxide ; S-Nitrosothiols ; SNACET
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2007
• 出版时间：15 October 2007
• 年：2007
• 卷：858
• 期：1-2
• 页码：32-41
• 全文大小：1002 K

文摘

Asymmetric dimethylarginine (ADMA), an endogenous 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 m/z 240 for DMA and m/z 246 for the internal standard (CD₃)₂NH 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 μM 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 μM. 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 HgCl₂, 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 μM, respectively. Oral administration of ¹⁵N-labelled SNACET to two healthy volunteers (1 μmol/kg) resulted in elevated urinary excretion of ¹⁵N-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.