The oxidized 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine (ox‐PAPC) products in human high‐density lipoproteins (HDLs) were investigated by low‐flow capillary electrophoresis‐mass ...spectrometry (low‐flow CE‐MS). To accelerate the optimization, native PAPC (n‐PAPC) standard was first analyzed by a commercial CE instrument with a photodiode array detector. The optimal separation buffer contained 60% (v/v) acetonitrile, 40% (v/v) methanol, 20 mM ammonium acetate, 0.5% (v/v) formic acid, and 0.1% (v/v) water. The selected separation voltage and capillary temperature were 20 kV and 23°C. The optimal CE separation buffer was then used for the low‐flow CE‐MS analysis. The selected MS conditions contained heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). No sheath gas was used for MS. The linear range for n‐PAPC was 2.5–100.0 µg/mL. The coefficient of determination (R2) was 0.9918. The concentration limit of detection was 1.52 µg/mL, and the concentration limit of quantitation was 4.60 µg/mL. The optimal low‐flow CE‐MS method showed good repeatability and sensitivity. The ox‐PAPC products in human HDLs were determined based on the in vitro ox‐PAPC products of n‐PAPC standard. Twenty‐one ox‐PAPC products have been analyzed in human HDLs. Uremic patients showed significantly higher levels of 15 ox‐PAPC products than healthy subjects.
The apolipoproteins (APOs) of human very low‐density lipoprotein (VLDL) were investigated by an optimized cyclodextrin‐micellar electrokinetic chromatography (CD‐MEKC) method. The separation buffer ...consisted of 20 mM sodium phosphate, 40 mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25 mM carboxymethyl‐β‐cyclodextrin (CM‐β‐CD) (pH 7.0). For CD‐MEKC separation, a sample injection time of 12 s, a separation voltage of 15 KV, and a capillary temperature of 15°C were chosen. The optimal CD‐MEKC method showed good resolution and repeatability for VLDL APOs. Identification and quantitation of VLDL APOs CI, CIII, and E were based on comparison with human APO standards. Good linear relationships with correlation coefficient (R2) 0.99 were obtained for APOs CI, CIII, and E standards. For these three APOs, the linear ranges were within 0.01‐0.54 mg/mL, and the concentration limits of detection (LODs) were lower than 0.02 mg/mL. Moreover, VLDL APOs from four uremic patients and four healthy subjects were compared. The uremic and healthy CD‐MEKC profiles showed dramatic difference. The levels of APO CIII were significantly higher for two patients, and the level of APO E was significantly higher for one patient. This study might be helpful for following the disease development of uremia and cardiovascular disease (CVD) in the future.
Surfactant‐coated multiwalled carbon nanotubes (MWNTs) were used as pseudostationary phase (PSP) in CE to investigate the total lipids of high‐density lipoproteins and low‐density lipoproteins. To ...optimize the CE conditions, several experimental factors including carbon nanotube concentration, bile salt concentration, sodium phosphate (PB) concentration, organic modifier concentration and buffer pH value have been examined. In addition, the CE capillary temperature and applied voltage have also been examined. The optimal separation buffer selected was a mixture of 3.2 mg/L MWNT, 50 mM bile salt, 10 mM PB, 20% 1‐propanol, pH 9.5. The optimal capillary temperature and applied voltage selected were 50°C and 20 kV, respectively. Phosphatidyl choline (PC) has been used as a model analyte and investigated by the optimal CE method. The linear range for PC was 0.1–3 mg/mL with a correlation coefficient of 0.9934, and the concentration LOD was 0.055 mg/mL. The optimal CE method has been used to characterize the total lipids of high‐density lipoprotein and low‐density lipoprotein. At absorbance 200 nm, one major peak and two or three minor peaks showed for the total lipids of lipoproteins within 13 minutes. Resolutions of the total lipids were enhanced using surfactant‐coated MWNTs as PSPs in the CE separation buffer. However, resolutions of the total lipids were not enhanced using surfactant‐coated single‐walled carbon nanotubes as PSPs in the CE separation buffer.
A simple liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI/MS) method has been developed to analyze oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) ...products on the lipoproteins of uremic patients. The native PAPC standard was in vitro oxidized by the Fenton reaction, and the ox-PAPC products were analyzed by LC- ESI/MS. For LC, a C8 column and a mobile phase (acetonitrile-isopropanol containing 0.1% formic acid (70:30, v/v)) were selected. For ESI/MS, the optimal conditions included sheath gas pressure (10 psi), capillary temperature (270 °C), and injection time (1000 ms). The identification of ox-PAPC products on human lipoproteins was based on the extracted ion chromatograms (EICs) and the ESI-MS spectra of the in vitro oxidation products of PAPC standard. The EICs and ESI-MS spectra showed good repeatability and sensitivity. A total of 21 ox-PAPC products was determined. Linear analysis has been performed for the phospholipid standard, 1, 2-Di-O-hexadecyl-sn-glycero-3-phosphocholine (PC(O-16:0/O-16:0)). The linear range was 5.0–100.0 µg/mL, and the coefficient of determination (R2) was 0.989. The concentration limit of detection (LOD) was 1.50 µg/mL, and the concentration limit of quantitation (LOQ) was 4.54 µg/mL. The selected 21 ox-PAPC products have been identified and quantified in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) of uremic and healthy subjects. Interestingly, the results showed that the levels of 18 products in VLDL, one product in LDL, and 19 products in HDL were significantly higher for uremic patients than healthy controls. This simple LC-ESI/MS method might accelerate the searching for biomarkers of uremia in the future.
A simple and fast micellar electrokinetic chromatography (MEKC) method was developed to investigate phospholipids isolated from human high‐density lipoproteins (HDL). To optimize the MEKC conditions, ...several factors including bile salt concentration and organic modifier concentration in the separation buffer as well as temperature have been examined. The optimal separation buffer chosen was a mixture of 50 mM bile salts, 30% v/v 1‐propanol and 10 mM sodium phosphate (pH 8.5). The applied voltage and temperature selected were 25 kV and 40°C, respectively. Meanwhile, high‐salt stacking has been performed for sample pre‐concentration to enhance peak sensitivity. Several factors including organic modifier concentration and salt concentration in the sample matrix as well as sample injection time have been optimized. The optimal sample buffer selected was a mixture of 100 mM NaCl and 20% 1‐propanol, and the optimal sample injection time selected was 32 s under a pressure of 0.5 psi. Several phospholipid standards including lysophosphatidyl choline, phosphatidyl choline (PC), sphingomyelin, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and phosphatidic acid have been studied using the optimal MEKC method. The MEKC profile of the mixed phospholipid standards showed good separation and reproducibility. The linear ranges for PC and sphingomyelin were 0.025–1.2 and 0.025–2.0 mg/mL, respectively. The concentration limits of detection of PC and sphingomyelin were 0.0156 and 0.0199 mg/mL, respectively. Using phosphatidic acid as an internal standard, precision and accuracy have been measured for PC and sphingomyelin. The intraday and interday quantitative analysis showed good results. The new MEKC method has been used to characterize native, in vitro oxidized and glycated human HDL phospholipids within 16 min. At absorbance 200 nm, two similar peaks were observed for native and oxidized HDL phospholipids, but three peaks were observed for glycated HDL phospholipids. Interestingly, at absorbance 234 nm, distinctively different MEKC profiles were observed for the three HDL phospholipids.
•A low-flow CE-MS method for analyzing ox-PAPC products was developed.•The low-flow CE-MS method showed good repeatability, linearity and sensitivity.•EIC and MS of in vitro oxidized PAPC were used ...to identify in vivo ox-PAPC.•A total of 21 ox-PAPC products were analyzed for human VLDLs.•Levels of 12 ox-PAPC products on uremic VLDLs were much higher than healthy VLDLs.
A simple and fast low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS) method has been developed to analyze oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human very low-density lipoproteins (VLDLs). Native PAPC standard was analyzed to optimize the low-flow CE-MS method. The optimal CE conditions included separation buffer (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 0.5% (v/v) formic acid, 20 mM ammonium acetate), sheath liquid (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 20 mM ammonium acetate), separation voltage (20 kV), separation capillary internal diameter (i.d.) (75 µm), separation capillary temperature (23˚C) and sample injection time (6 s). The selected MS conditions included heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). Sheath gas was not used in this study. The total ion chromatograms (TICs), extracted ion chromatograms (EICs) and MS spectra of native PAPC standard and its in vitro oxidation products showed good repeatability and sensitivity. To determine the ox-PAPC products in human VLDLs, the EICs and MS spectra of VLDLs were compared with the in vitro oxidation products of native PAPC standard. For native PAPC standard, the measured linear range was 2.5 - 100.0 µg/mL, and the coefficients of determination (R2) was 0.9994. The concentration limit of detection (LOD) was 0.44 µg/mL, and the concentration limit of quantitation (LOQ) was 1.34 µg/mL. A total of 21 ox-PAPC products were analyzed for the VLDLs of healthy and uremic subjects. The levels of 7 short-chain and 5 long-chain ox-PAPC products on uremic VLDLs were significantly higher than healthy VLDLs. This simple low-flow CE-MS method might be a good alternative for LC-MS for the analysis of ox-PAPC products. Furthermore, it might also help scientists to expedite the search for uremic biomarkers.
C-Reactive protein (CRP) is a clinical biomarker of inflammation, and high levels of CRP correlate with cardiovascular disease. The objectives of this study were to test our hypothesis that oxidized ...low-density lipoprotein (ox-LDL) induces the release of CRP from human aortic endothelial cells (HAECs) and to optimize several analytical methods to identify CRP released from cultured cells in a model of atherogenic stress. HAECs were incubated with copper-oxidized LDL, and the supernatant was subsequently purified by diethylaminoethyl chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified an optimal buffer for the elution of CRP, which contained 0.05 M sodium phosphate and 2.0 M NaCl (pH 4.5). Purified CRP was digested with trypsin and subjected to high-performance LC with an optimal mobile phase of acetonitrile–water containing 0.1% formic acid (50:50, v/v) and an optimal mobile phase flow rate of 0.2 mL/min. We identified optimal parameters for MS/MS analysis of CRP, including sheath gas pressure (80 psi), capillary temperature (275 °C), collision energy (25%), tube lens offset (−5 V), auxiliary gas pressure (0 psi), and isolation width of parent ion (m/z value = 3). Characterization of CRP was based on the extracted ion chromatograms and selected multiple-reaction monitoring spectra of three peptides (peptide-1, -2, and -3) derived from trypsin-digested intact CRP standard. CRP peptide-2 and peptide-3 were identified in the supernatant of ox-LDL-treated HAECs. Confirmation of CRP was based on LC-MS/MS and enzyme-linked immunosorbent assay analysis of CRP in purified HAEC supernatant, as well as real-time PCR analysis of CRP mRNA levels in HAECs.
•A new CD-MEKC method for analyzing human HDL apolipoproteins (apos) was developed.•Apos AI, AII, CI and CIII were identified in human HDL using apo standards.•The new CD-MEKC method showed good ...repeatability, linearity and sensitivity.•The CD-MEKC profiles of healthy subjects and uremic patients differed significantly.
A cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method has been developed to determine the apolipoproteins (apos) of human high-density lipoprotein (HDL). The optimal CD-MEKC conditions included a separation buffer mixture of 5 mM sodium phosphate, 40 mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25 mM carboxymethyl-β-CD (CM-β-CD) and pH 7.0. The separation voltage was 15 kV, and the capillary temperature was 15℃. The CD-MEKC profiles of human HDL apolipoproteins showed good repeatability and sensitivity.
Linear analysis has been performed for human apolipoprotein standards including apos AI, AII, CI, CII, CIII and E. Linear regression lines with coefficients of determination (R2) greater than 0.99 were obtained for apos AI, AII, CI, CII and E. The linear ranges for the six apolipoproteins were within 0.18–0.70 mg/mL, and the concentration limits of detection (LOD) were lower than 0.0617 mg/mL.
Apos AI, AII, CI and CIII were identified and quantified in human HDL by comparing with apolipoprotein standards. Furthermore, the CD-MEKC profiles of uremic patients differed significantly from healthy subjects. The concentration ratios of apo AI/apo CIII were significantly lower for uremic patients than healthy subjects.
This study demonstrated the feasibility of determining human HDL apolipoproteins by CD-MEKC. In the future, it might help monitor the progression of uremia and cardiovascular disease.
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.
