Böhmer Anke, Gambaryan Stepan, Flentje Markus, Jordan Jens, Tsikas Dimitrios
Institute of Clinical Pharmacology, Hannover Medical School, 30623 Hannover, Germany.
Institute of Clinical Biochemistry, University of Wuerzburg, Wuerzburg, Germany; Sechenov Institute of Evolutionary Physiology, Russian Academy of Sciences, S. Petersburg, Russia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Aug 15;965:173-82. doi: 10.1016/j.jchromb.2014.06.025. Epub 2014 Jun 28.
In healthy human subjects, less than 0.2% of l-arginine is converted to l-citrulline and nitric oxide (NO) by NO synthases (NOS), a metabolic pathway present in all cell types. Assessment of NOS activity in vitro and in vivo by measuring l-citrulline or NO is difficult. l-citrulline is formed from l-arginine to a much higher extent by other pathways including the urea cycle. Furthermore, NO is a very short-lived gaseous molecule and is oxidized to nitrite and nitrate which are ubiquitous. In fact, nitrite and nitrate are also derived from food and air and are major laboratory contaminants. Further, NOS (in the uncoupled state) are also able to produce superoxide in addition and/or instead of l-citrulline and NO. The difficulties of NOS assays based on l-citrulline and NO measurement can only in part be overcome by sophisticated techniques including use of radio-labeled ((3)H or (14)C) and stable-isotope labeled ((15)N2 at the guanidine group) l-arginine analogs as substrates for NOS and measurement of radio-labeled l-citrulline and (15)N-labeled nitrite and nitrate, respectively. In the present work, we report on the development, validation and application of an UPLC-MS/MS method for the assessment of the activity of recombinant NOS enzymes by using [guanidino-(15)N2]-l-arginine (20 μM for recombinant NOS, 5mM in cell systems) as the substrate and by measuring [ureido-(15)N]-l-citrulline as the reaction product (usually formed at concentrations below 1 μM) using (2)H7-l-citrulline as the internal standard. The lower limit of detection of the method is about 80 fmol (2)H7-l-citrulline. In cell systems, exceeding [guanidino-(15)N2]-l-arginine is removed by strong cation exchanger solid-phase extraction. The method was cross-validated by a GC-MS assay that measures simultaneously (15)N-nitrite and (15)N-nitrate as pentafluorobenzyl derivatives, with unlabeled nitrite and nitrate serving as the internal standards. By means of this UPLC-MS/MS (15)N-citrulline assay, N(G)-nitro-arginine (100 μM) was found to inhibit recombinant inducible NOS (iNOS) activity (by 38%), whereas nitrite and GSSG (each at 500 μM) did not affect iNOS activity at all. Nitrite and GSSG at pathophysiological concentrations are unlikely to uncouple NOS. NOS activity was not detectable in platelets of healthy humans by the UPLC-MS/MS and GC-MS assays.
在健康人体中，一氧化氮合酶（NOS）可将不到0.2%的L-精氨酸转化为L-瓜氨酸和一氧化氮（NO），这是一条存在于所有细胞类型中的代谢途径。通过测量L-瓜氨酸或NO在体外和体内评估NOS活性具有一定难度。L-瓜氨酸可通过包括尿素循环在内的其他途径从L-精氨酸大量生成。此外，NO是一种寿命极短的气体分子，会被氧化为普遍存在的亚硝酸盐和硝酸盐。事实上，亚硝酸盐和硝酸盐也来源于食物和空气，是实验室的主要污染物。此外，（处于解偶联状态的）NOS还能够产生超氧化物，取代和/或补充L-瓜氨酸和NO的生成。基于L-瓜氨酸和NO测量的NOS检测方法所面临的困难，只能部分地通过复杂技术来克服，这些技术包括使用放射性标记（（3）H或（14）C）和稳定同位素标记（胍基处为（15）N2）的L-精氨酸类似物作为NOS的底物，并分别测量放射性标记的L-瓜氨酸和（15）N标记的亚硝酸盐和硝酸盐。在本研究中，我们报告了一种超高效液相色谱-串联质谱（UPLC-MS/MS）方法的开发、验证及应用，该方法以[胍基-（15）N2]-L-精氨酸（重组NOS用20μM，细胞系统用5mM）为底物，以（2）H7-L-瓜氨酸为内标，通过测量[脲基-（15）N]-L-瓜氨酸作为反应产物（通常以低于1μM的浓度生成）来评估重组NOS酶的活性。该方法的检测下限约为80飞摩尔（2）H7-L-瓜氨酸。在细胞系统中，过量的[胍基-（15）N2]-L-精氨酸通过强阳离子交换剂固相萃取去除。该方法通过气相色谱-质谱（GC-MS）检测进行交叉验证，GC-MS检测同时测量作为五氟苄基衍生物的（15）N-亚硝酸盐和（15）N-硝酸盐，未标记的亚硝酸盐和硝酸盐作为内标。通过这种UPLC-MS/MS（15）N-瓜氨酸检测方法发现，N（G）-硝基精氨酸（100μM）可抑制重组诱导型NOS（iNOS）活性（抑制38%），而亚硝酸盐和谷胱甘肽二硫化物（GSSG，均为500μM）对iNOS活性完全没有影响。病理生理浓度的亚硝酸盐和GSSG不太可能使NOS解偶联。通过UPLC-MS/MS和GC-MS检测在健康人的血小板中未检测到NOS活性。
