Investigação de malondialdeído livre e total por eletroforese capilar visando a análise de amostras de urina e plasma

Abstract

Malondialdehyde (MDA) is the most widely used biomarker of lipid peroxidation, and the development of selective, reliable, and rapid methods for its determination in biological samples is essential. Thus, the present work aimed to develop and optimize a Capillary Zone Electrophoresis with Direct UV Detection (CZE – UV) method for the determination of the MDA biomarker in urine samples from individuals tested for COVID-19 and in plasma samples from individuals supplemented with omega-3. The electrophoretic mobility of MDA was determined for the first time, which proved to be of paramount importance for the development of the CZE – UV method. Implementing an effective mobility x pH curve and simulation in the PeakMaster® software allowed the determination of the most appropriate experimental conditions for the analysis. Thus, the optimized background electrolyte (BGE) comprised TRIS 40 mmol L-1 , HCl 20 mmol L-1 , and CTAB 2.5 mmol L-1 (pH 8.0). It was verified that MDA presents good absorption in the UV region. Thus, it was monitored at 266 nm. Under these conditions, MDA was detected in approximately 1 min, so this would be the method for determining MDA in biological samples by Capillary Electrophoresis (CE) with the shortest migration time ever developed. It was not possible to detect the free MDA signal in the urine and plasma samples, even by implementing a preconcentration of the analyte using the fieldamplified sample injection (FASI) technique. The Box-Behnken experimental design was applied to optimize preconcentration. The factors evaluated in this experimental design were the BGE concentration, the voltage applied during the electrokinetic injection, and the time of the electrokinetic injection. To determine total MDA, bound MDA was released into free MDA by an alkaline hydrolysis. However, no MDA signal could be detected in the plasma sample. The same hydrolysis procedure in urine samples allowed the detection of an MDA signal, which was confirmed by fortification with the same. Quantification of total MDA by implementing a matrix-matched calibration curve minimized the matrix effect. Total MDA was quantified in 75 urine samples from individuals tested for COVID-19, and these values were normalized with urinary creatinine. However, the MDA concentrations found were very high compared to studies found in the literature. Thus, the following studies involving sample treatment could be performed to help understand the high MDA values found in urine samples and promote method improvement: study of MDA stability in urine after alkaline hydrolysis, measurement of total MDA in urine samples by the thiobarbituric acid reactive substances (TBARS) assay, which is the most widely used method for this purpose, and extraction of MDA after alkaline hydrolysis with a suitable solvent, to avoid the influence of interferents.