In vitro oxidized and glycated human low-density lipoprotein particles characterized by capillary zone electrophoresis.

A simple capillary zone electrophoresis (CZE) method was used to determine native, in vitro Cu(2+) and glucose modified low-density lipoprotein (LDL) particles for four healthy subjects. The LDL electropherograms are highly reproducible with good precisions of effective mobility and peak area. The native LDL capillary electrophoresis (CE) profile shows a major peak with lower mobility and two minor peaks with higher mobilities. For three-hour Cu(2+) oxidation, one major peak with mobility close to that of the native major peak, and one minor peak with mobility extending to -47 x 10(-5)cm(2)V(-1)s(-1) appear. For eighteen-hour Cu(2+) oxidation, one major peak with mobility much higher than that of the native major peak appears. As the reaction time for LDL and Cu(2+) increases from 0 to 24h, effective mobility of the LDL major peak increases, suggesting that LDL particles become more negatively charged and oxidized as the time increases. The in vitro glycated LDL particles are characterized by a major peak and two minor peaks. Mobility of the major peak is close to that of native major peak, but the second minor peak is much more negatively charged with mobility extending to -53 x 10(-5)cm(2)V(-1)s(-1). Native, oxidized and glycated LDL particles show distinctive differences in their CZE profiles. Agarose electrophoresis shows that the charge to mass ratios of native, three-hour Cu(2+) and glucose modified LDL particles are similar, but that of eighteen-hour Cu(2+) oxidized LDL particles is higher.

Characterization of in vitro modified human high-density lipoprotein particles and phospholipids by capillary zone electrophoresis and LC ESI-MS.

A simple capillary zone electrophoresis (CZE) method was used to characterize native, in vitro oxidized and glycated human high-density lipoprotein (HDL) particles. Both native and in vitro oxidized HDL capillary electrophoresis (CE) profiles showed a major peak, but the oxidized HDL particles had higher effective mobilities. The in vitro glycated HDL particles showed a major peak and one or two minor peaks. The effective mobility of the major peak of glycated HDL was similar to that of the major peak of native HDL, whereas the effective mobilities of the two minor peaks were much lower. For the analysis of HDL phospholipids, a solid phase extraction procedure was optimized and a LC ESI-MS method was developed. Several possible HDL phospholipid molecular species including phosphatidylcholine (PC 16:0/18:2, 16:0/18:1, 18:0/18:2 and 18:0/18:1), sphingomyelin (SM 16:0) and lyso-phosphatidylcholine (lysoPC 16:0 and 18:0) were found. It appeared that the ion intensity ratios of hydroperoxy-PC or epoxyhydroxy-PC (16:0/hydroperoxy-18:2 or 16:0/epoxyhydroxy-18:2, m/z 790.4) and trihydroxy-PC (16:0/trihydroxy-18:2, m/z 808.3) relative to PC (C16:0/C18:2, m/z 758.5) were higher for oxidized HDL than for native and glycated HDL. It should be helpful to use both CZE and LC ESI-MS methods for analyzing high-density lipoproteins from patients of cardiovascular disease. Their combination may be also useful for further studies concerning the role of oxidized and glycated HDLs in the development of atherosclerosis.

Separation and determination of in vitro oxidized phospholipids by capillary zone electrophoresis.

A simple capillary zone electrophoresis (CZE) method was used to determine in vitro oxidized phosphatidyl choline (ox-PC). To optimize the capillary electrophoresis (CE) conditions, organic buffer additives, buffer ionic strength, buffer pH, and applied voltage were examined. The optimal CE separation buffer chosen was an aqueous-organic solvent system containing 10% sodium phosphate buffer (5 mM, pH 7.40), 80% methanol, and 10% acetonitrile. One major peak with a small shoulder was found for phosphatidyl choline (PC), whereas one major peak and a complex region containing several lower-mobility peaks were found for ox-PC. The lower-mobility species of ox-PC has high levels of conjugated dienes characterized by strong absorbance at 234 nm. The electropherograms of PC and ox-PC were significantly different and highly reproducible. The intensities of lower-mobility species decreased significantly when the antioxidant vitamin C concentration was increased from 6 to 600 microM. This study provides a simple CZE method to differentiate in vitro oxidized from nonoxidized PC molecular species.