Open Tubular Capillary Electrochromatography: Technique for Oxidation and Interaction Studies on Human Low-Density Lipoproteins
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- 作者：Ruth Kuldvee ; Lucia D'ulivo ; Gebrenegus Yohannes ; Petrus W. Lindenburg ; Minna Laine ; Katariina Ö ; ö ; rni ; Petri Kovanen ; and Marja-Liisa Riekkola
- 刊名：Analytical Chemistry
- 出版年：2006
- 出版时间：April 15, 2006
- 年：2006
- 卷：78
- 期：8
- 页码：2665 - 2671
- 全文大小：267K
- 年卷期：v.78,no.8(April 15, 2006)
- ISSN：1520-6882
文摘
A novel, open tubular capillary electrochromatographicmethod was developed for the in vitro oxidation of low-density lipoprotein (LDL) particles. Low-density lipoprotein particles with molar mass of ~2.5 MDa yielded astable stationary phase at temperatures 25 and 37 
C andat pH values from 3.2 to 7.4. The quality of the coatingswas not influenced by variations in the LDL concentrationin 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 electronmicroscopy, employed to shed light on the location andcoating density of LDL particles on the inner surface ofthe capillary wall, confirmed the presence of an LDLmonolayer and almost 100% coating efficiency (99 ± 8%).In addition, the radioactivity measurements allowed estimation of the amount of LDL present in a single capillarycoating. Capillaries coated with human LDL particles weresubmitted to different oxidative conditions by changingthe concentration of the oxidant (CuSO4), oxidation time,pH value, and temperature. The oxidation procedure wasfollowed with electroosmotic flow mobility, which servedas an indicator of the increase in total negative charges ofLDL coatings, and by asymmetrical field flow fractionation,which measured the changes in size of the lipoproteinparticles. The results indicated that oxidation of LDL wasprogressing with increasing time, temperature, and concentration of the oxidant as expected. The oxidationprocess was faster around neutral pH values (pH 6.5-7.4) and inhibited at acidic pH values (pH 5.5 and lower).
C andat pH values from 3.2 to 7.4. The quality of the coatingswas not influenced by variations in the LDL concentrationin 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 electronmicroscopy, employed to shed light on the location andcoating density of LDL particles on the inner surface ofthe capillary wall, confirmed the presence of an LDLmonolayer and almost 100% coating efficiency (99 ± 8%).In addition, the radioactivity measurements allowed estimation of the amount of LDL present in a single capillarycoating. Capillaries coated with human LDL particles weresubmitted to different oxidative conditions by changingthe concentration of the oxidant (CuSO4), oxidation time,pH value, and temperature. The oxidation procedure wasfollowed with electroosmotic flow mobility, which servedas an indicator of the increase in total negative charges ofLDL coatings, and by asymmetrical field flow fractionation,which measured the changes in size of the lipoproteinparticles. The results indicated that oxidation of LDL wasprogressing with increasing time, temperature, and concentration of the oxidant as expected. The oxidationprocess was faster around neutral pH values (pH 6.5-7.4) and inhibited at acidic pH values (pH 5.5 and lower).