Open Tubular Capillary Electrochromatography:  Technique for Oxidation and Interaction Studies on Human Low-Density Lipoproteins

Abstract

A novel, open tubular capillary electrochromatographic method was developed for the in vitro oxidation of low-density lipoprotein (LDL) particles. Low-density lipoprotein particles with molar mass of approximately 2.5 MDa yielded a stable stationary phase at temperatures 25 and 37 degrees C and at pH values from 3.2 to 7.4. The quality of the coatings was not influenced by variations in the LDL concentration in the coating solutions (within the range of 2-0.015 mg/mL) with the coating procedure used in the study. Radiolabeled LDL stationary phases and scanning electron microscopy, employed to shed light on the location and coating density of LDL particles on the inner surface of the capillary wall, confirmed the presence of an LDL monolayer and almost 100% coating efficiency (99 +/- 8%). In addition, the radioactivity measurements allowed estimation of the amount of LDL present in a single capillary coating. Capillaries coated with human LDL particles were submitted to different oxidative conditions by changing the concentration of the oxidant (CuSO4), oxidation time, pH value, and temperature. The oxidation procedure was followed with electroosmotic flow mobility, which served as an indicator of the increase in total negative charges of LDL coatings, and by asymmetrical field flow fractionation, which measured the changes in size of the lipoprotein particles. The results indicated that oxidation of LDL was progressing with increasing time, temperature, and concentration of the oxidant as expected. The oxidation process was faster around neutral pH values (pH 6.5-7.4) and inhibited at acidic pH values (pH 5.5 and lower).

Coating of open tubular capillaries with discoidal and spherical high‐density lipoprotein particles in electrochromatography

“…The coating densities for spherical and discoidal HDL were lower than those we obtained earlier for LDL and VLDL particles [14,15]. The results, nevertheless, indicate that HDL coating is stable and robust, and that piranha acid cannot remove all the particles from the capillary.…”

“…In most of the studies reported in the literature, successful analysis of lipoproteins has been achieved only after permanent [12] or dynamic [13] coating of the capillary wall has been applied to suppress adsorption of the particles. Adopting a quite different approach, we have recently demonstrated the usefulness of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particle coatings in lipoprotein oxidation studies by CEC [14,15].…”

Recent progress in polar stationary phases for CEC

“…The results indicated that the strength of the interactions between steroids and the LDL coating increased with hydrophobicity of the drug, and the EOF of the acetylated LDL coating increased evidently than that of unmodified LDL. Furthermore, both radiolabeled LDL stationary phases and SEM were employed to clarify the location and coating density of LDL particles on the inner surface of the capillary wall [21]. The results confirmed the presence of an LDL monolayer and almost 100% coating efficiency (99 6 8%).…”

“…Additionally, a method to evaluate electrochromatographic retention of charged analytes in o-CEC was presented, which combines o-CEC and flow counterbalanced CE. The affinity between the immobilized RNA aptamer and its two target molecules, FMN and FAD, was characterized at different Mg 21 concentrations.…”

Recent applications in CEC

“…Kuldvee et al [36,37] used inverse OT-CEC to study the reactions and interactions of low-density lipoproteins (LDLs). Etched fused-silica capillaries were coated with native or acetylated LDL simply by flushing with an appropriate LDL solution.…”