Development of Analytical Methods for the Determination of Methylarginines in Serum

Abstract

Nitric oxide (NO) plays a crucial role in numerous physiological pathways including the regulation of the endothelium that lines blood vessels throughout the body. Therefore, in order to maintain good endothelial health, there must be a careful homeostasis of NO. Under pathological conditions that impair the production of NO, endothelial function is disrupted which can result in various pathologies including cardiovascular diseases (CVDs) and respiratory disorders. A class of endogenous compounds that inhibit the enzyme responsible for NO synthesis in vivo are the methylated arginines (MAs). Given their propensity for attenuating NO production, it comes as no surprise that MAs have been implicated in several diseases. Increased blood concentrations of asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and monomethylarginine (MMA) have been reported in patients suffering from CVDs. However, despite evidence demonstrating the link between MAs and these diseases, no diagnostic concentrations have yet been established. The goal of this work was to develop an analytical method capable of rapidly determining the concentrations of MAs in blood samples so that threshold concentrations indicative of disease could be established. Further efforts were then made to fabricate a point-of-care device that could be used in a clinical setting to measure MAs as a means of preventative diagnostics. Analyzing components in a serum sample is a very challenging endeavor because of the incredible complexity of the sample matrix. To alleviate matrix interferents, a method was developed to rapidly isolate MAs from serum using a newly developed heating procedure. The sample was immersed in a boiling water bath which caused it to solidify. Solvent was then added to the congealed serum and briefly homogenized to permit solid-liquid extraction to take place. After a brief incubation period at room temperature, the sample was centrifuged to sediment the aggregated serum proteins, leaving the small molecules of interest in the supernatant. The supernatant was then derivatized with naphthalene-2,3-dicarboxaldehyde to label the MAs for analysis by capillary electrophoresis (CE) with fluorescence detection. A CE method was developed using sulfobutylether-b-cyclodextrin and dimethylsulfoxide as buffer modifiers to obtain good resolution between the MAs and the other components in serum-derived samples. Under optimized conditions, baseline resolution was achieved which allowed precise quantitation of the MAs. The separation method was then transferred to a microchip electrophoresis (MCE) device that made it possible to perform the same analysis more rapidly on a smaller, portable device. MAs were separated using this MCE platform as a first step towards the development of a point-of-care device to perform clinical analyses on-chip.