Chairman Students |
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Name: Perpetua E. Tan
Enrolled: 1991 - 1996 Origin: Philippines
Supervisor: Dr. John Matthew Low Title of Thesis: Studies on Plasma Catecholamines in Man (Analytical Techniques and Applications) Outline of the Thesis: Catecholamines are hormones naturally present in the human body. They are important for assessing the activity of the sympathetic nervous system. Low nanomolar levels of catecholamines, however, demand a sensitive, reproducible and reliable method. This thesis include the development of an analytical technique for measuring the levels of catecholamines (mainly noradrenaline and adrenaline) in plasma. The thesis also includes application of the developed technique to clinical research studies. A small part of the thesis was also aimed at setting up a protein binding method to analyze the binding of noradrenaline and adrenaline in plasma. The methodology involved extraction of the catecholamines onto acid-washed alumina under basic condition. The alumina was washed with water and the catecholamines and internal standard were finally re-absorbed into dilute acetic acid. The acid extract was lyophilized and the residue was re-dissolved using the HPLC mobile phase and the filtrate was then injected into the HPLC system for separation on an Ultrasphere IP-C18 reversed phase column. The catecholamines were monitored by a coulometric detector. 2ml of plasma was required for the analysis. The method was linear from 25 pg/ml to 3000 pg/ml. The limit of detection was at 25 pg/ml. The absolute recovery was found to range between 60% to 75% for noradrenaline, adrenaline and dihydroxybenzylamine. The recovery of noradrenaline and adrenaline relative to the internal standard was between 97% to 106%. The intraassay coefficient of variation was between 0.98% and 6.87% while the interassay coefficient of variation was between 3.12% and 12.56%. This method was applied on samples in two research studies under the Department. The first study's objective was to compare the maternal cardiovascular and catecholamine response after rapid sequence induction of anaesthesia in patients undergoing caesarian section. The patients were divided into two groups, with one group receiving Thiopentone and the other group receiving Propofol as inducting agent respectively. In neonates, umbilical blood gas, oxygen content and catecholamine concentrations were also determined to compare the neonatal outcome between the two groups. The maternal concentrations of noradrenaline and adrenaline in both groups increased after tracheal intubation. Similar trends were observed with their arterial pressures. Propofol was found to be more effective in attenuating the hypertensive and the catecholamine response associated with tracheal intubation and laryngoscopy. But there was no significant difference in the neonatal outcome between the two groups. The second study's objective was to determine the intravascular absorption of adrenaline and the circulating levels of both noradrenaline and adrenaline during functional endoscopic sinus surgery. There was marked increase in the adrenaline levels that reached its peak value within 4 minutes after commencing infiltration. Adrenaline levels returned to basal levels within 5 minutes after the peak adrenaline was recorded. Absence of significant activation of the sympathetic nervous system that could affect the measured adrenaline levels was demonstrated by a relatively constant noradrenaline concentration. An attempt to establish a method for plasma protein binding analysis of catecholamines was performed. Several factors were taken into consideration to optimize the binding conditions. These included the type of preservative used to prevent degredation of catecholamines, dialysis buffer. Dialysis time, dialyzing membrane, and appropriate catecholamine concentrations. However, even with optimization of these factors, the results obtained did not meet conditions for method acceptability. Hence, further tests were needed to improve the drug binding analytical technique.
Publications arising from her thesis: (A) Scientific Papers:
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