الملخص الإنجليزي
Background: Atherosclerosis is an inflammatory disease of the arterial wall which is the major cause of cardiovascular disease worldwide. The disease involves the formation of plaques in arterial walls restricting blood flow and increasing the risk of myocardial infarction. It is now believed that atherosclerosis represents a state of increased oxidative stress mainly characterized by lipid oxidation in the vascular wall. Atherosclerosis is mainly initiated by an accumulation and subsequent oxidation of low density lipoprotein (LDL) particles in the arterial Intima which is triggered by circulating free radicals. Lipid peroxidation plays a major role in the development of atherosclerosis. However, the oxidation damage caused by free radicals can be minimized by many antioxidants. Recently bilirubin was highlighted to have antioxidant effects in vitro and in vivo, The lipoprotein oxidizability is usually studied by the isolation of single lipoprotein (LDL) fractions, which is a tedious time-consuming process which renders it unuseful for clinical routine practice. Recently limited reports showed that the use of unfractionated serum may be of similar value.
Aims: This project had four main aims: 1) unfractionated serum was tested for its possible use to study serum oxidizability in vitro by formation of a typical oxidation curve consisting of the following components: basal oxidation status value (BOS), lag phase, propagation phase and saturation phase. 2) The antioxidant effect of different concentrations of commercial bilirubin was investigated on the components of the serum oxidation curve. 3) Bilirubin levels were measured in patients with acute coronary syndrome and controls, and its correlation with BOS and metabolic syndrome components was investigated. 4) In addition, we attempted to downsize the routine cuvette procedure, by which only a limited number of samples could be tested for serum oxidation status, to a plate method involving 96 samples and less sample in one run, in an effort to maximize the ability to test samples routinely for basal serum oxidation status.
Materials and Methods: 24uL of serum was added to a solution of PBS containing 100uM copper (total volume of the reaction mixture is 1.2 ml) in a standard quartz cuvette. The formation of a typical lipid oxidation curve was monitored by spectrophotometry at 245 nm using a standard UV- spectrophotometer. Secondly, the copper induced serum oxidation was measured spectophotometrically at 245 nm in the presence of different bilirubin concentrations (2-128uM) to test for the antioxidant effect of bilirubin. Thirdly, serum total bilirubin in acute coronary syndrome patients and controls was quantitated by using cobas 11 lanalyzer (Roche Diagnostics). Also BOS for the same samples were measured spectrophotometry after copper induced oxidation. Fourthly, the procedure was downsized by using a 96 well quartz plate, were only 3ul of serum sample and 150ul of copper reagent was used and the plate was measured spectrophotometry at 245 nm in one run in a plate reader machine. SPSS 10 package was used for statistical analysis.
Results and-Discussion: A typical lipid oxidation curve was obtained using copper as an -oxidizing agent..on unfractionated serumandbilirubin was shown to significantly inhibit
serum oxidation with increasing concentrations. Bilirubin levels were significantly decreased in ACS patients compared to the controls, but no significant correlation was found between bilirubin levels and.BOS in the patients or controls. Importantly bowever, increased bilirubin . : levels correlated positively with LDL size, consistent with the less atherogenic profile of the controls as larger LDL particles are less atherogenic than in the patients, and correlated negatively with ApoB, a proposed cardiovascular risk factor, findings that are consistent with bilirubin's role as an antioxidant. On the other hand, BOS was higher in ACS patients and showed a negative correlation with LDL size, HDL-C, ApoAl and a positive association with the rest of the parameters of metabolic syndrome which is consistent with an atherogenic profile of the patients. Multiple linear regression analysis revealed that serum triglycerides were the main independent predictors of BOS in the patients reflecting that increased TG levels is a major determinant of basal oxidation status . Interestingly also, there was a significant positive association of BOS with liver enzymes, particularly GGT which is an established serum oxidant linked to cardiovascular disease. Finally, results obtained by the modified plate procedure for measuring BOS showed results comparable to the standard cuvette method; findings that indicate promising use of this method in routine clinical practice, however, further modifications and standardization of the new technique are required.
