Isolation Of High-density Lipoprotein Research Articles

Identification of proteins associated with apolipoprotein A-I-containing lipoproteins purified by selected-affinity immunosorption.

The isolation of apolipoprotein A-I-containing lipoproteins [Lp(A-I)] by selected-affinity immunosorption minimizes the loss of associated proteins that occurs during the isolation of high-density lipoproteins (HDL) by sequential ultracentrifugation. We have used two-dimensional gel electrophoretic analysis to separate the proteins associated with Lp(A-I). Using a combination of amino acid sequencing of transblotted proteins and Western blotting with specific antisera, we have identified a number of associated proteins. The positions of the apolipoproteins (apo) A-I, A-II, A-IV, C-III, D, and E were located on the gels. Lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein were identified in association with Lp(A-I) to a greater extent than found associated with HDL after centrifugation. In addition to those proteins previously identified in association with HDL, we detected a number of plasma proteins associated with Lp(A-I), namely, fibrinogen, haptoglobin, proline-rich protein (C4b-binding protein), and apolipoprotein J (SP40,40 sulfated glycoprotein). The co-isolation of these proteins with Lp(A-I) does not appear to be an artifact in that they have very low affinity for a sham column containing covalently bound preimmune goat IgG in place of the anti-apoA-I IgG. These findings suggest that in addition to apolipoproteins that exist largely in association with lipoproteins there is another class of proteins which exist in lipoprotein-associated form and in the dispersed state. Detection and identification of these lipoprotein-associated proteins may aid in the mechanistic determination of a number of observed functions attributed to HDL.

Six methods for isolating high-density lipoprotein compared, with use of the reference method for quantifying cholesterol in serum.

Using 90 serum specimens, we compared six routine procedures for high-density lipoprotein (HDL) isolation to determine the biases, if any, of each. Use of the Reference Method for cholesterol (Duncan et al., Centers for Disease Control, Atlanta, GA) and automated dispensing equipment helped ensure the accuracy of the cholesterol measurements and minimized errors from sample and reagent manipulations. Regression analysis of the results showed significant differences between most HDL isolation methods, except for those involving precipitation with heparin-MnCl2 (1.0 mol/L) or polyethylene glycol 6000, which yielded comparable results with a slope close to one and a zero intercept. The dextran sulfate (Mr 500 000)-MgCl2 method had the largest proportional and constant bias with respect to those two methods. All the methods produced comparable results in the clinically important low HDL-cholesterol range (250 to 350 mg/L), but biases were significant at high concentrations. We conclude that these increased biases in the upper ranges of HDL-cholesterol concentrations are the result of increased heterogeneity of HDL and the different mechanisms involved in forming the insoluble complexes between lipoproteins and the various precipitation reagents.

Isolation of high density lipoproteins from rat intestinal epithelial cells.

Previous studies have defined forms of high density lipoproteins (HDL) in rat mesenteric lymph, suggesting that they have a secretory origin. This study describes the isolation and characterization of intestinal intracellular HDL. Two preparations were made as follows: (a) Rat enterocytes were isolated and a Golgi organelle fraction was prepared. (b) Cell homogenates were subjected to nitrogen cavitation and a cytoplasmic fraction was prepared. Lipoproteins were isolated from both preparations by sequential ultracentrifugation. When the HDL fraction (1.07-1.21 g/ml) was subjected to isopyknic density gradient ultracentrifugation, a peak of apoproteins A-I and B (apoA-I and apoB, respectively) was found at a density of 1.11-1.14 g/ml. Electron microscopy of the fraction showed spherical particles ranging in size from 6 to 13 nm. Immunoelectrophoresis revealed a precipitin arc in the alpha region against apoA-I which extended into the pre-beta region where a precipitin arc against apoB was also seen. ApoB antisera depleted the pre-beta particles whereas the alpha migrating particles remained. Lipid analysis of the whole HDL fraction revealed phospholipid, cholesteryl ester, and triglyceride as the major lipids. [3H]leucine was then administered into the duodenum and a radiolabeled intracellular HDL fraction was isolated. The newly synthesized apoproteins of the HDL fraction, as determined by gel electrophoresis, were apoB, apoA-I, and apolipoprotein A-IV (ApoA-IV). Immunoprecipitation of the apoB particles revealed apoA-I and apoA-IV in the supernatant. These data demonstrate that there are at least two intracellular intestinal forms of HDL particles, one of which contains apoB. The other particle contains apoA-I and apoA-IV, has alpha mobility, is spherical, and resembles a particle found in the lymph.

Isolation of high-density lipoproteins by immunoaffinity column chromatography from hog plasma

1. 1. High density lipoprotein (HDL) was isolated from hog plasma by a simple immunoaffinity column chromatography procedure using immobilized anti-apolipoprotein AI. 2. 2. The composition of HDL isolated by immunoaffinity chromatography was nearly identical to that of a control sample that was isolated by an alternate method utilizing ultracentrifugation and gel chromatography. 3. 3. The HDL isolated by immunoaffinity chromatography had a larger number of polypeptide components than the control as indicated by acrylamide gel electrophoresis in the presence of urea. 4. 4. When the HDL isolated by immunoaffinity chromatography was applied to a heparin-agarose column the amount of protein retained was approximately twice that of the control. These findings indicate that the ultracentrifugation procedure probably induced the loss of apolipoprotein E containing components from the HDL complex.

Catabolism of the apolipoproteins of HDL in control and nephrotic rats

125I-labeled high density lipoprotein (HDL) from control rats, or from rats made nephrotic by puromycin amino-nucleoside, was injected into control or nephrotic rats. At 5 and 20 h, the amount and distribution of label remaining in apolipoproteins HDL, A-I, E, A-IV and the C apolipoproteins was measured after ultracentrifugal isolation of HDL and SDS-polyacrylamide gel electrophoretic separation of each apolipoprotein. There were no significant differences in the removal rates of apolipoprotein HDL or of the individual apolipoproteins when the removal of HDL of controls was compared to HDL of nephrotics. HDL from nephrotic rats contains less than 10% of either the apolipoprotein A-IV or apolipoprotein E content of control HDL, indicating that neither apolipoprotein A-IV nor apolipoprotein E play a significant role in determining the catabolic fate of rat HDL. In severely nephrotic animals the apolipoprotein C content of HDL was reduced to 50% of control values and the apolipoprotein A-I content of HDL rose to 87% of the total apolipoprotein. The individual apolipoproteins of HDL from either nephrotics or controls were catabolized at the same rates irrespective of the degree of nephrosis or altered HDL apolipoprotein composition. The apparent fractional catabolic rates for apolipoprotein HDL and for each of the apolipoproteins, determined after 20 h, did not differ from one another, and all were reduced by half in the nephrotic rats compared to the normal controls. These results support the concept that HDL is catabolized as a particle mediated by apolipoprotein A-I recognition, and they reinforce earlier work indicating that increased synthesis is the dominant factor responsible for increased plasma HDL concentrations in experimental nephrosis.

Improved method for determination of high-density-lipoprotein cholesterol I. Isolation of high-density lipoproteins by use of polyethylene glycol 6000.

We describe a modified method of precipitating low- and very-low-density lipoproteins with polyethylene glycol, for quantitation of high-density lipoprotein cholesterol. A 100 g/L concentration of polyethylene glycol 6000 in a buffered (pH 10) solution is used. Under these conditions precipitation of beta-lipoproteins is complete. Values for HDL cholesterol as measured with this method fully correspond to those obtained on separating the lipoproteins by ultracentrifugation or with heparin-Mn2+, 46 mmol/L; are slightly higher than those obtained with heparin-Mn2+, 92 mmol/L; and are significantly higher than those determined after precipitation with polyethylene glycol, 100 g/L at pH 8.0. Comparison with results by the ultracentrifugation method showed excellent correlation (r = 0.957) with a good correspondence of results (slope 1.065 and y-intercept -22.231); comparison with the heparin-Mn2+, 46 mmol/L, method gave r = 0.998 with slope of 0.985 and y-intercept 2.963. The method is unaffected by precipitation and centrifugation time and temperature (up to 24 h with 4-22 degrees C temperature for precipitation and between 10 and 30 min with 4-22 degrees C temperature for centrifugation). This extremely simple method is particularly suitable for routine analyses of many samples, even in small laboratories.