Abstract
Increased asymmetric dimethylarginine (ADMA) in human plasma has been associated with reduced generation of nitric oxide, leading to atherosclerotic diseases. ADMA may therefore be an important biomarker for cardiovascular disease. In the present study, three sample preparation techniques were compared regarding the quantification of L-arginine and ADMA in human plasma: (A) protein precipitation (PP) based on aqueous trichloroacetic acid (TCA), (B) PP using a mixture of ammonia and acetonitrile, and (C) solid-phase extraction (SPE). The samples were analysed by using high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The analytical performance of (A) was comparable with that of (C), demonstrating recoveries of >90%, coefficient of variations (CVs, %) of <8, and a resolution (Rs) between ADMA and symmetric dimethylarginine (SDMA) of 1.2. (B) was disregarded due to recoveries below 75%. (A) was validated with good results regarding linearity (>0.994), precision (<5%), and sensitivity (lower limit of quantification (LLOQ)) of 0.14 µM and 12 nM for L-arginine and ADMA, respectively. Due to the simplicity and speed of procedure (A), this approach may serve as preferred sample preparation of human plasma samples before HPLC-FLD in providing important information regarding elevated ADMA concentrations.
Highlights
Nitric oxide (NO) is important in numerous biological processes including the relaxation of smooth muscles and inhibition of platelet aggregation [1]. e endothelial nitric oxide synthase enzyme is involved in mediating vasodilation in vascular endothelium due to its role in the conversion of L-arginine to L-citrulline and NO, the latter inducing vasodilation
Three sample preparation techniques were compared regarding the quantification of L-arginine and asymmetric dimethylarginine (ADMA) in human plasma: (A) protein precipitation (PP) based on aqueous trichloroacetic acid (TCA), (B) PP using a mixture of ammonia and acetonitrile, and (C) solid-phase extraction (SPE). e samples were analysed by using high-performance liquid chromatography with fluorescence detection (HPLC-FLD). e analytical performance of (A) was comparable with that of (C), demonstrating recoveries of >90%, coefficient of variations (CVs, %) of 0.994), precision (
Increased concentrations of ADMA have been found to correlate with impaired endothelial vasodilation in several clinical studies possibly leading to the development of atherosclerotic diseases [3,4,5], and ADMA is considered to be an important biomarker. e similar NG,NG′-dimethyl-Larginine, SDMA, is not an inhibitor of endothelial nitric oxide synthase (eNOS) but competes with L-arginine in cellular uptake [6]. e reliability of ADMA as a biomarker of endothelial dysfunction is dependent on its precise analytical quantification, which is challenging due to the narrow concentration range found in healthy individuals [7]
Summary
Nitric oxide (NO) is important in numerous biological processes including the relaxation of smooth muscles and inhibition of platelet aggregation [1]. e endothelial nitric oxide synthase (eNOS) enzyme is involved in mediating vasodilation in vascular endothelium due to its role in the conversion of L-arginine to L-citrulline and NO, the latter inducing vasodilation. A sample preparation step is often required before chromatographic analysis in order to separate the analytes of interest from proteins and other possible interferences in plasma [8]. A few publications exist for Journal of Analytical Methods in Chemistry quantification of L-arginine, ADMA, and SDMA, where simple PP has been performed with 5-aminosalicylic acid [14], ethanol [15], or acetonitrile/ammonia [16]. These methods still consist of several steps including lyophilisation or evaporation
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