Development, validation and application of analytical methods to measure prognostic biomarkers in patients receiving on-pump coronary artery grafting surgery (2013)
Temporary interception and then re-infusion of oxygenated blood into heart during on-pumpCoronary Artery Bypass Grafting (CABG) surgery causes ischemia-reperfusion injury (IRI).Propofol is an intravenous anesthetic agent that maybe potentially cardioprotective against IRI. Thisthesis presents the development, validation and application of analytical methods for monitoringpropofol and a series of prognostic biomarkers in hope of identifying contributory factors to IRI andpropofol cardioprotection.I developed a Capillary Electrophoresis (CE) method to quantify propofol concentrations inblood. Propofol concentrations in the μg/mL range were measured from 400 μL samples. A dosefindingstudy using this method determined a practical infusion rate of 120 μg•kg•min⁻¹ to achievethe target blood concentration of 5 μg•mL⁻¹.To measure the oxidative stress biomarker, 15-F₂t₋isoprostane, the nitrosative stressbiomarker, 3-nitrotyrosine, the myocardial protective factor, adenosine, and the cardiovascular riskfactor, asymmetric dimethylarginine (ADMA), I developed simple, sensitive and robust LiquidChromatography-Mass Spectrometry (LC-MS) or Liquid Chromatography Tandem MassSpectrometry (LC-MS/MS) methods. Briefly, a basic mobile phase and base-resistant column wereused for the LC-MS analysis of 15-F₂t-isoprostane. A one-step solid phase extraction andpentafluorophenyl (PFP) core-shell column were employed for the LC-MS/MS analysis of 3-nitrotyrosine. A Strong Cation Exchange (SCX) solid phase extraction and modifier-free mobilephase were implemented for the LC-MS/MS analysis of adenosine. A new derivatization method toenable baseline separation of ADMA and its regio-isomer symmetric dimethylarginine (SDMA) wasdeveloped for the ADMA LC-MS/MS quantitation. Performance parameters for these methods,including linearity, precision, accuracy, Limit of Detection (LOD), Limit of Quantitation (LOQ),Lower Limit of Quantitation (LLOQ) and stability were found satisfactory.Concentrations of 15-F₂t₋isoprostane, 3-nitrotyrosine and adenosine were found to rise afteron-pump CABG surgery. However, these changes were not able to explain the cardioprotectiveeffect of propofol. Nonetheless, the correlations of 15-F₂t₋isoprostane with diabetes, glucoseconcentration and PTEN level were significant. Patients with low cardiac output syndromeexperienced more 3-nitrotyrosine increase than patients without this syndrome. The basal adenosinelevel was found to increase more in patients with low left ventricular ejection fraction. Thesefindings and the underlying methodologies are important for identifying new prognostic biomarkers.