Separation Of High-density Lipoprotein Research Articles

On-line miniaturized asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry with selected reaction monitoring for quantitative analysis of phospholipids in plasma lipoproteins

A direct analytical method for high speed quantitative analysis of lipids in human blood plasma using on-line chip-type asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry (cAF4-ESI-MS/MS) with selected reaction monitoring (SRM) is described in this study. Utilizing a miniaturized cAF4 channel, high speed size separation of high density lipoproteins (HDL) and low density lipoproteins (LDL) from plasma samples can be accomplished at a microflow rate along with simultaneous desalting of lipoproteins, both of which are conducive to direct ESI of lipids in lipoproteins. This study demonstrates that the SRM method to monitor phospholipids during cAF4-ESI-MS/MS can be successfully applied to the quantitation of lipid molecules in plasma lipoproteins without the need of a separate lipid extraction process. For quantitation of lipids in HDL and LDL during cAF4-ESI-MS/MS runs, a protein standard (carbonic anhydrase, 29kDa) was added to each plasma sample as an internal standard such that a peak intensity of y67+5 ions, which are high abundant SRM product ions of CA, could be utilized to calculate the relative intensity of each lipid molecule. The developed method was applied to plasma samples from 10 patients with coronary artery disease (CAD) and 10 healthy control samples, and quantitative analysis of 39 lipid molecules including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylglycerols, and phosphatidylinositols, resulted in the selection of 13 PL species showing more than 2.5 fold difference in relative abundance (p<0.01) between the groups. The present study demonstrates a high speed analytical method for determining plasma lipid content and distribution without an organic solvent extraction of lipids from plasma.

Separation of Apolipoprotein A‐I from Human Plasma by On‐Line Two Dimensional Liquid Chromatography

On‐line two dimensional liquid chromatography (2D LC) was used to determine apolipoprotein A‐I (apo A‐I) in human plasma without sample pretreatment. The water soluble apolipoproteins were separated from other plasma proteins and lipids by reversed phase (RP) LC in the first dimension using acetonitrile/20 mM aqueous ammonium acetate as mobile phase. A strong anion exchange (SAX) column was subsequently employed in the second dimension to separate the apo A‐I in the apolipoprotein fraction using pH gradient elution with simple buffer mixtures of ammonium acetate and ammonium formate as mobile phase. Compared to other methods requiring separation of high density lipoprotein (HDL) fraction by ultracentrifugation and delipidation of apolipoproteins with organic solvents, loss of apo A‐I is avoided in the on‐line 2D approach. The concentration of apo A‐I in plasma, determined by using the calibration curve for commercial apo A‐I, was found to be 1.12 mg/mL. The identity of apo A‐I isolated from human plasma was confirmed by trypsination and peptide mass fingerprinting (PMF).

Separation of high-density lipoproteins into apolipoprotein E-poor and apolipoprotein E-rich subfractions by fast protein liquid chromatography using a heparin affinity column

The aim of this paper is to describe a new methodology for the separation of human high-density lipoproteins (HDL) into apolipoprotein (apo) E-poor and apo E-rich subfractions by fast protein liquid chromatography (FPLC) using a heparin affinity column. Recoveries for apolipoproteins AI, AII, CI, CII, CIII, and E were 68.9, 74.7, 71.9, 73.5, 40.0, and 55.8%, respectively. We provide suggestive evidence that apo E-rich HDL is produced from apo E-poor HDL by the displacement of apo AI by apo E. Apo E-poor HDL was the predominant fraction. The molar ratio of apo E to apo AI in apo E-poor HDL was 0.02 and 0.01 for the subjects studied while in apo E-rich HDL it was 1.86 and 1.25. The molar ratios of the C apolipoproteins to apo AI are markedly different between the subfractions.

Automated enzymatic standardized lipid analyses for plasma and lipoprotein fractions

Excellent normal ranges for plasma lipid and lipoprotein cholesterol levels have been developed by the Lipid Research Clinics program, standardized by the Centers for Disease Control (CDC). However these values were generated by methods not currently in use in most clinical chemistry laboratories. Automated enzymatic methods for cholesterol, triglycérides and free glycerol determinations, as well as a dextran sulfate-Mg 2+ procedure for separation of high density lipoproteins (HDL) with standardization are described. Similar methods for the measurement of unesterified cholesterol and phospholipids are also given. Serum pools for total cholesterol with values ranging from 1220–3490 mg/l produced coefficients of variation (CV) ⩽2.85%; reference values for low total cholesterol samples in a range of 280–727 mg/l gave CV of 4.35% or less; HDL cholesterol reference values of 265–640 mg/l yielded CV ⩽3.70%; and values for triglycerides between 0.74 and 3.05 mmol/l gave CV of 4.22% or less through three to eight testing cycles (9–24 mth). These data indicate that long term CDC standardization of total cholesterol, triglycerides, and HDL cholesterol can be obtained with automated enzymatic methods utilizing commercially available reagents.

A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor

A method was developed for the separation of the high density lipoprotein subclasses HDL2 and HDL3 from human serum. Six serum samples are fractionated in a single-step ultracentrifugal procedure using the Beckman (SW-40) swinging bucket rotor. The method is based on a difference in flotation rate of the high density lipoprotein subclasses. Separation of HDL2 and HDL3 is accomplished by a discontinuous NaBr density gradient applied on top of 2 ml of serum brought to a density of 1.40 g/ml. After centrifugation, high density lipoprotein subclass profiles were obtained using a specially designed gradient fractionator. Contamination of the isolated high density lipoprotein subclasses by serum albumin or by apolipoprotein B-containing lipoproteins was minimal while only a slight overlap between the HDL2 and HDL3 profiles was observed. Chemical and immunochemical analyses of the high density lipoprotein subclasses isolated by the present method were in close agreement with the results obtained by rate-zonal density gradient ultracentrifugation in zonal rotors (Patsch, et al. 1980. J. Biol. Chem. 255: 3178-3185). The major advantage of the method presented in this paper as compared with the zonal rotor method is the possibility to analyze as many as six serum samples simultaneously.

The application of sodium deoxycholate and Sephacryl-200 for the delipidation and separation of high density lipoprotein.

A method to remove lipids from human plasma high-density lipoprotein (HDL) was developed. The procedure required column chromatography on Sephacryl-200 in the presence of the bile salt sodium deoxycholate. The lipid free protein obtained retained the immunological properties of the native HDL.

Loss of A-apoprotein immunoreactivity during high-density lipoprotein separation.

By use of an electroimmunoassay, concentrations of A-apoproteins were estimated in serum and in corresponding apoprotein fractions isolated by ultracentrifugation. These values were compared with high-density lipoprotein concentrations determined by analytical ultracentrifugation. Concentrations of A-apoproteins estimated in serum were considerably higher than in isolated high-density lipoprotein fractions. These discrepancies could not be accounted for entirely by material losses into other fractions during ultracentrifugal fractionation. No comparable differences in apoprotein-B concentrations were observed during the ultracentrifugal separation of low-density lipoprotein. Concentrations of A-apoproteins estimated in the residual serum after precipitation of low-density lipoproteins by heparin and manganous ions were also lower than in the corresponding whole sera. The discrepancies persisted after treatment of serum and isolated fractions with tetramethylurea, urea (9 mol/l), and by heating at 52 degrees C for 3 hours. It is considered that separation by ultracentrifugation induces subtle alterations in the surface structure of the lipoprotein species which give rise to changes in immunoreactivity.

Fractionation of Egg Yolk Lipoproteins by Rate Zonal Ultracentrifugation

Crude high density lipoprotein of egg yolk was fractionated by rate zonal ultracentrifugation. Rate zonal ultracentrifugation was carried out with zonal rotor RPZ-43T (660ml) in density gradient d. 1.00-1.40 by sodium bromide at 40000rpm.By the ultracentrifugation for four hours, the separation of high density lipoprotein (HDL) from low density lipoprotein (LDL) was observed, but HDL was completely purified only by the second ultracentrifiugation for twenty four hours after the first procedure.The purified HDL shows only one peak in the schlieren pattern of the analytical ultracentrifugation.