Characterization of high density lipoprotein subspecies: structural studies by single vertical spin ultracentrifugation and immunoaffinity chromatography

Affinity columns containing anti-apolipoprotein A-I or A-II were used to fractionate plasma into subpopulations of lipoprotein particles containing: a) apoA-I [Lp(A-I)], b) apoA-I and A-II [Lp(A-I with A-II)], and c) apoA-I but no A-II [Lp(A-I without A-II)]. Single vertical spin and electron microscopy analyses of these HDL subpopulations demonstrated that acid elution from the affinity columns caused no detectable change in size and density of the three subpopulation particles. Analysis by nondenaturing gradient gel electrophoresis of the three subpopulations found in four normal subjects identified nine HDL subspecies, designated [1] through [9] in order of increasing size; [3-7] were the major subspecies. Lp(A-I with A-II) is composed primarily of subspecies [3],[5], and [6], and may contain some subspecies [2] and [7], while Lp(A-I without A-II) represents mainly [4] and [7] and the minor subspecies [1],[2],[8], and [9]. HDL subspecies [4],[5], and [6] are found in the standard sequential flotation density cuts for both HDL3 and HDL2, which illustrates the limitations of the latter terminology. Using single vertical spin ultracentrifugation, HDL fractions were located and isolated for physical and chemical analyses, including immunoassay for apoA-I, A-II, and C-II. The distribution of the Lp(A-I without A-II) particles corresponded closely to the apoC-II distribution. An apoA-I-rich, cholesteryl ester- and phospholipid-poor subspecies was identified in the dense HDL fractions. HDL subspecies [7] was found to contain at least three separate subspecies designated [7a], [7b], and [7c]. Based on these and previously published results (Brouillette, C. G., et al. 1984. Biochemistry. 23: 359-367), we propose that the HDL subspecies adjacent in size generally differ by the association/lack of association of a hinge-like domain of amphipathic helixes in a single molecule of apoA-I.     

Characterization of apoA-I-containing lipoprotein subpopulations secreted by HepG2 cells

Recent immunoaffinity studies demonstrate two populations of high density lipoprotein (HDL) particles: one contains both apolipoprotein (apo) A-I and A-II [Lp(A-I w A-II)], and the other contains apoA-I but no A-II [Lp(A-I w/o A-II)]. To investigate whether these two populations are derived from different precursors, we applied sequential immunoaffinity chromatography to study the lipoprotein complexes in HepG2 conditioned serum-free medium. The apparent secretion rates of apoA-I, A-II, E, D, A-IV, and lecithin:cholesterol acyltransferase (LCAT) were 4013 +/- 1368, 851 +/- 217, 414 +/- 64, 171 +/- 51, 32 +/- 14, and 2.9 +/- 0.7 ng/mg cell protein per 24 h, respectively (n = 3-5). Anti-A-II removed all apoA-II but only 39 +/- 5% (n = 5) apoA-I from the medium. These HepG2 Lp(A-I w A-II) also contained 31 +/- 1% (n = 5) of the apoD and 82 +/- 2% (n = 3) of the apoE in the medium. The apoE existed both as E and E-A-II complex. Lipoproteins isolated from the apoA-II-free medium by anti-A-I contained, besides apoA-I, 60 +/- 3% of the medium apoD and trace quantities of apoE. The majority of HepG2 apoA-IV (78 +/- 4%) (n = 3) and LCAT (85 +/- 6%) (n = 3) was not associated with either apoA-I or A-II. HepG2 Lp(A-I w A-II) contained relatively more lipids than Lp(A-I w/o A-II) (45 vs. 37%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations

Two populations of apolipoprotein (apo) A-I-containing lipoprotein particles are found in high density lipoproteins (HDL): those that also contain apo A-II[Lp(A-I w A-II)] and those that do not [Lp(A-I w/o A-II)]. Lp(A-I w/o A-II) comprised two distinct particle sizes with mean hydrates Stokes diameter of 10.5 nm for Lp(A-I w/o A-II)1 and 8.5 nm for Lp(A-I w/o A-II)2. To study the effect of ultracentrifugation on these particles, Lp(A-I w/o A-II) and Lp(A-I w A-II) were isolated from the plasma and the ultracentrifugal HDL (d 1.063-1.21 g/ml fractions) of five normolipidemic and three hyperlipidemic subjects. The size subpopulations of these particles were studied by gradient polyacrylamide gel electrophoresis. Several consistent differences were detected between plasma Lp(A-I w/o A-II) and HDL Lp(A-I w/o A-II). First, in all subjects, the relative proportion of Lp(A-I w/o A-II)1 to Lp(A-I w/o A-II)2 isolated from HDL was reduced. Second, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 were considerably reduced in HDL. Third, a distinct population of particles with approximate Stokes diameter of 7.1 nm usually absent in plasma was detected in HDL Lp(A-I w/o A-II). Little difference in subpopulation distribution was detected between Lp(A-I w A-II) isolated from the plasma and HDL of the same subject. When plasma Lp(A-I w/o A-II) and Lp(A-I w A-II) were centrifuged, 14% and 4% of A-I were, respectively, recovered in the D greater than 1.21 g/ml fraction. Only 2% A-II was found in this density fraction. These studies show that the Lp(A-I w/o A-II) particles are less stable than Lp(A-I w A-II) particles upon ultracentrifugation. Among the various Lp(A-I w/o A-II) subpopulations, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 are most labile.     

Distribution and localization of lecithin:cholesterol acyltransferase and cholesteryl ester transfer activity in A-I-containing lipoproteins

Two types of A-I-containing lipoproteins are found in human high density lipoproteins (HDL): particles with A-II (Lp(A-I with A-II] and particles without A-II (Lp(A-I without A-II]. We have studied the distribution of lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer (CET) activities in these particles. Lp(A-I with A-II) and Lp(A-I without A-II) particles were isolated from ten normolipidemic subjects by anti-A-I and anti-A-II immunosorbents. Most plasma LCAT mass (70 +/- 15%), LCAT (69 +/- 16%), and CET (81 +/- 15%) activities were detected in Lp(A-I without A-II). Some LCAT (mass: 16 +/- 7%, activity: 17 +/- 8%) and CET activities (7 +/- 8%) were detected in Lp(A-I with A-II). To determine the size subspecies that contain LCAT and CET activities, isolated Lp(A-I with A-II) and Lp(A-I without A-II) particles of six subjects were further fractionated by gel filtration column chromatography. In Lp(A-I without A-II), most LCAT and CET activities were associated with different size particles, with the majority of the LCAT and CET activities located in particles with hydrated Stokes diameters of 11.6 +/- 0.4 nm and 10.0 +/- 0.6 nm, respectively. In Lp(A-I with A-II), most of the LCAT and CET activities were located in particles similar in size: 11.1 +/- 0.4 nm and 10.6 +/- 0.3 nm, respectively. Ultracentrifugation of A-I-containing lipoproteins resulted in dissociation of both LCAT and CET activities from the particles. Furthermore, essentially all CET and LCAT activities were recovered in the non-B-containing plasma obtained by anti-LDL immunoaffinity chromatography. This report, therefore, provides direct evidence for the association of LCAT and CET protein with A-I-containing lipoproteins. Our conclusions pertain to fasting normolipidemic subjects and may not be applicable to hyperlipidemic or nonfasting subjects.     

Identification and characterization of rat serum lipoprotein subclasses. Isolation by chromatography on agarose columns and sequential immunoprecipitation

Lipoproteins, present in serum of chow-fed rats, were fractionated according to size by chromatography of serum on 6% agarose columns. The distributions of apolipoprotein (apo) A-I, E, and A-IV within the high density lipoprotein (HDL) size range (i.e., lipoprotein complexes smaller than low density lipoproteins) showed the existence of lipoprotein subclasses with different size and chemical composition. Sequential immunoprecipitations were performed on these fractions obtained by agarose column chromatography, using specific antisera against apoA-I, apoE, and apoA-IV. The resulting precipitates and supernatants were analyzed for cholesteryl esters, unesterified cholesterol, phospholipids, triglycerides, and specific lipoproteins. The following conclusions were drawn from these experiments. Sixty-three +/- 3% of apoE in the total HDL size range is present on a large particle (mol wt 750,000). This lipoprotein contains apoE as its sole protein constituent and is called LpE. Thirty-nine +/- 4% of the cholesterol found in the HDL size range is present in this fraction. The cholesterol:phospholipid ratio is 1:1.1. Sixty-nine +/- 8% of apoA-I in the total HDL size range is present on a smaller particle (mol wt 250,000). This apoA-I-HDL has apoA-I as its major protein component and possibly contains minor amounts of C apoproteins and A-II, but neither apoE nor apoA-IV. It contains 39 +/- 8% of the total cholesterol found in the HDL size range and the cholesterol:phospholipid ratio is 1:1.6.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation in the human apolipoprotein A-I gene. Dominant effect on the level and characteristics of plasma high density lipoproteins

Epidemiologic and genetic data suggest an inverse relationship between plasma high density lipoprotein (HDL) cholesterol and the incidence of premature coronary artery disease. Some of the defects leading to low levels of HDL may be a consequence of mutations in the genes coding for HDL apolipoproteins A-I and A-II or for enzymes that modify these particles. A proband with plasma apoA-I and HDL cholesterol that are below 15% of normal levels and with marked bilateral arcus senilis was shown to be heterozygous for a 45-base pair deletion in exon four of the apoA-I gene. This most likely represents a de novo mutation since neither parent carries the mutant allele. The protein product of this allele is predicted to be missing 15 (Glu146-Arg160) of the 22 amino acids comprising the third amphipathic helical domain. The HDL of the proband and his family were studied. Using anti-A-I and anti-A-II immunosorbents we found three populations of HDL particles in the proband. One contained both apoA-I and A-II, Lp(A-I w A-II); one contained apoA-I but no A-II, Lp(A-I w/o A-II); and the third (an unusual one) contained apoA-II but no A-I. Only Lp(A-I w A-II) and (A-I w/o A-II) were present in the plasma of the proband's parents and brother. Analysis of the HDL particles of the proband by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two protein bands with a molecular mass differing by 6% in the vicinity of 28 kDa whereas the HDL particles of the family members exhibited only a single apoA-I band. The largely dominant effect of this mutant allele (designated apoA-ISeattle) on HDL levels suggests that HDL particles containing any number of mutant apoA-I polypeptides are catabolized rapidly.     

Protein transfer between A-I-containing lipoprotein subpopulations: evidence of non-transferable A-I in particles with A-II.

Transfer of apolipoproteins (apo) between the two subpopulations of apo A-I-containing lipoproteins in human plasma: those with A-II [Lp(AI w AII)] and those without [Lp(AI w/o AII)], were studied by observing the transfer of 125I-apo from a radiolabeled subpopulation to an unlabeled subpopulation in vitro. When Lp(AI w AII) was directly radioiodinated, 50.3 +/- 7.4 and 19.5 +/- 7.7% (n = 6) of the total radioactivity was associated with A-I and A-II, respectively. In radioiodinated Lp(AI w/o AII), 71.5 +/- 6.8% (n = 6) of the total radioactivity was A-I-associated. Time-course studies showed that, while some radiolabeled proteins transferred from one population of HDL particles to another within minutes, at least several hours were necessary for transfer to approach equilibrium. Incubation of the subpopulations at equal A-I mass resulted in the transfer of 51.8 +/- 5.0% (n = 4) of total radioactivity from [125I]Lp(AI w/o AII) to Lp(AI w AII) at 37 degrees C in 24 h. The specific activity (S.A.) of A-I in the two subpopulations after incubation was nearly identical. Under similar incubation conditions, only 13.4 +/- 4.6% (n = 4) of total radioactivity was transferred from [125I]Lp(AI w AII) to Lp(AI w/o AII). The S.A. of A-I after incubation was 2-fold higher in particles with A-II than in particles without A-II. These phenomena were also observed with iodinated high-density lipoproteins (HDL) isolated by ultracentrifugation and subsequently subfractionated by immunoaffinity chromatography. However, when Lp(AI w AII) radiolabeled by in vitro exchange with free [125I]A-I was incubated with unlabeled Lp(AI w/o AII), the S.A. of A-I in particles with and without A-II differed by only 18% after incubation. These data are consistent with the following: (1) in both populations of HDL particles, some radiolabeled proteins transferred rapidly (minutes or less), while others transferred slowly (hours); (2) when Lp(AI w AII) and Lp(AI w/o AII) were directly iodinated, all labeled A-I in particles without A-II were transferable, but some labeled AI in particles with A-II were not; (3) when Lp(AI w AII) were labeled by in vitro exchange with [125I]A-I, considerably more labeled A-I were transferable. These observations suggest the presence of non-transferable A-I in Lp(AI w AII).     

Apolipoprotein A-II inhibits high density lipoprotein remodeling and lipid-poor apolipoprotein A-I formation

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

Lipoprotein lipase and hepatic lipase: their relationship with HDL subspecies Lp(A-I) and Lp(A-I,A-II)

HDL subspecies Lp(A-I) and Lp(A-I,A-II) have different anti-atherogenic potentials. To determine the role of lipoprotein lipase (LPL) and hepatic lipase (HL) in regulating these particles, we measured these enzyme activities in 28 healthy subjects with well-controlled Type 1 diabetes, and studied their relationship with Lp(A-I) and Lp(A-I,A-II). LPL was positively correlated with the apolipoprotein A-I (apoA-I), cholesterol, and phospholipid mass in total Lp(A-I), and with the apoA-I in large Lp(A-I) (r >or= 0.58, P >or= 0.001). HL was negatively correlated with all the above Lp(A-I) parameters plus Lp(A-I) triglyceride (r >or= -0.53, P or= 0.50, P 相似文献   

In vivo metabolism of apolipoprotein A-I on high density lipoprotein particles LpA-I and LpA-I,A-II.

Apolipoprotein (apo) A-I is the major protein in high density lipoproteins (HDL) and is found in two major subclasses of lipoproteins, those containing apolipoprotein A-II (termed LpA-I,A-II) and those without apoA-II (termed LpA-I). The in vivo kinetics of apoA-I on LpA-I and LpA-I,A-II were investigated in normolipidemic human subjects. In the first series of studies, radiolabeled apoA-I and apoA-II were reassociated with autologous plasma lipoproteins and injected into normal subjects. LpA-I and LpA-I,A-II were isolated from plasma at selected time points by immunoaffinity chromatography. By 24 h after injection, only 52.8 +/- 1.0% of the apoA-I in LpA-I remained, whereas 66.9 +/- 2.7% of apoA-I in LpA-I,A-II remained (P less than 0.01). In the second series of studies, purified apoA-I was labeled with either 131I or 125I and reassociated with autologous plasma. Isolated LpA-I and LpA-I,A-II particles differentially labeled with 131I-labeled apoA-I and 125I-labeled apoA-I, respectively, were simultaneously injected into study subjects. The plasma residence time of apoA-I injected on LpA-I (mean 4.39 days) was substantially shorter than that of apoA-I injected on LpA-I,A-II (mean 5.17 days), with a mean difference in residence times of 0.79 +/- 0.08 days (P less than 0.001). These data demonstrate that apoA-I injected on LpA-I is catabolized more rapidly than apoA-I injected on LpA-I,A-II. The results are consistent with the concept that LpA-I and LpA-I,A-II have divergent metabolic pathways.     

In vivo metabolism of apolipoproteins A-IV and A-I associated with high density lipoprotein in normolipidemic subjects

The kinetics of apolipoprotein A-IV associated with high density lipoproteins (HDL) of plasma from fasting human subjects was followed for 15 days in five healthy normolipidemic volunteers. Purified apoA-IV and apoA-I were radioiodinated, respectively, with 125I and 131I, incubated in vitro with normal HDL, isolated at density 1.250 g/ml, and finally reinjected intravenously as HDL-125I-labeled apoA-IV and HDL-131I-labeled apoA-I. Blood samples were withdrawn at regular intervals for 15 days, and 24-h urine samples were collected. More than 93% (93.5 +/- 0.9%) of apoA-IV was recovered in apoA-I-containing lipoprotein particles after affinity chromatography on an anti-apoA-I column and 69.7 +/- 4.8% was bound to apoA-II in apoA-I:A-II particles separated on an anti-apoA-II column. 125I-labeled apoA-IV showed a much faster decay than 131I-labeled apoA-I for the first 5 days and thereafter the curves became parallel. Urinary/plasma ratios (U/P) for the 125I-labeled parallel. Urinary/plasma ratios (U/P) for the 125I-labeled apoA-IV were much higher than those for 131I-labeled apoA-I for the first days, but the U/P curves became parallel for the last 7 days, suggesting heterogeneity of apoA-IV metabolism. A heterogeneous multicompartmental model was constructed to describe the metabolism of lipoprotein particles containing apoA-IV and apoA-I and to calculate the kinetic parameters, fitting simultaneously all plasma and urine data for both tracers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of A-I-containing lipoproteins in subjects with A-I Milano variant

The A-I Milano variant of apolipoprotein A-I (A-IM), by virtue of its Arg-173----Cys substitution, is capable of forming a disulfide bond with the 77-amino-acid apolipoprotein A-II polypeptide (A-IIS) as well as with itself to produce dimers, A-IM/A-IIS and A-IM/A-IM, respectively. A-I-containing lipoproteins (Lp): particles with A-II (Lp(A-I with A-11)) and particles without A-II (Lp(A-I without A-II)) in the plasma of two nonhyperlipidemic A-IM carriers were investigated to determine the effect of A-IM on these lipoproteins. Despite the existence of abnormal apolipoprotein dimers and the unusually low HDL cholesterol (17 and 14 mg/dl), A-I (67 and 75 mg/dl), and A-II (18 and 18 mg/dl) levels in the two carriers, the plasma A-I of the carriers was distributed between Lp(A-I with A-II) and Lp(A-I without A-II) in a proportion comparable to that observed in normals. As expected, A-IM/A-IIS mixed dimer was found in carrier Lp(A-I with A-II). However, A-IM/A-IM dimer was located almost exclusively in carrier Lp(A-I without A-II). Chemical (dimethylsuberimidate) crosslinking of the protein moieties of the major subpopulations of Lp(A-I with A-II) and Lp(A-I without A-II) of normal and A-IM carriers showed that Lp(A-I with A-II), which is located predominantly in the 7.8-9.7 nm interval ((HDL2a + 3a + 3b)gge), had an apparent protein molecular weight equivalent to two molecules of A-I and one to two molecules of A-II per particle. Most of the Lp(A-I without A-II) particles, located predominantly in the size intervals of 9.7-12.9 nm (designated (HDL2b)gge) and 8.2-8.8 nm (HDL3a)gge) had protein moieties exhibiting a molecular weight equivalence predominantly of four and three molecules of A-I, respectively. A small quantity of particles with apparent protein content of two molecules of A-I in the 7.2-8.2 nm interval ((HDL3b + 3c)gge) was also detected. These studies showed that in nonhyperlipidemic A-IM carriers, the occurrence of apolipoprotein dimers had not markedly affected the protein stoichiometry of Lp(A-I with A-II) and Lp(A-I without A-II).     

Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II

Two populations of A-I-containing lipoprotein particles: A-I-containing lipoprotein with A-II (Lp (A-I with A-II], and A-I-containing lipoprotein without A-II (Lp (A-I without A-II] have been isolated from plasma of 10 normolipidemic subjects by immunoaffinity chromatography and characterized. Both types of particles possess alpha-electrophoretic mobility and hydrated density in the range of plasma high-density lipoproteins (HDL). Lp (A-I without A-II) and Lp (A-I with A-II) are heterogeneous in size. Lp (A-I without A-II) comprised two distinct particle sizes with mean apparent molecular weight and Stokes diameter of 3.01 X 10(5), and 10.8 nm for Lp (A-I without A-II)1, and 1.64 X 10(5), and 8.5 nm for Lp (A-I without A-II)2. Lp (A-I with A-II) usually contained particles of at least three distinct molecular sizes with mean apparent molecular weight and Stokes diameter of 2.28 X 10(5) and 9.6 nm for Lp (A-I with A-II)1, 1.80 X 10(5) and 8.9 nm for Lp (A-I with A-II)2, and 1.25 X 10(5) and 8.0 nm for Lp (A-I with A-II)3. Apoproteins C, D, and E, and lecithin:cholesterol acyltransferase (LCAT) were detected in both Lp (A-I without A-II) and Lp (A-I with A-II) with most of the apoprotein D, and E, and LCAT (EC 2.3.1.43) in Lp (A-I with A-II) particles. Lp (A-I without A-II) had a slightly higher lipid/protein ratio than Lp (A-I with A-II). Lp (A-I with A-II) had an A-I/A-II molar ratio of approximately 2:1. The percentage of plasma A-I associated with Lp (A-I without A-II) was highly correlated with the A-I/A-II ratio of plasma (r = 0.96, n = 10). The variation in A-I/A-II ratio of HDL density subfractions therefore reflects different proportions of two discrete types of particles: particles containing A-I and A-II in a nearly constant ratio and particles containing A-II but no A-II. Each type of particle is heterogeneous in size and in apoprotein composition.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

Distribution of apolipoprotein A-IV among lipoprotein subclasses in rat serum

The distribution of apolipoproteins (apo) A-I, A-IV, and E in sera of fed and fasted rats was studied using various methods for the isolation of lipoproteins. Serum concentrations of apoA-I and apoA-IV decreased significantly during fasting (16 and 31%, respectively), while apoE concentrations remained essentially the same. Chromatography of sera on 6% agarose columns showed that apoA-IV is present on HDL and as so-called "free" apoA-IV. The concentration of "free" apoA-IV decreased six- to seven-fold during fasting, explaining the decrease in total serum apoA-IV. Serum apoA-I and apoE are almost exclusively associated with HDL-sized particles. When sera are centrifuged at a density of 1.21 g/ml, marked quantities of apoA-I (8-9%) and apoE (11-22%) are recovered in the "lipoprotein-deficient" infranatant, suggesting that ultracentrifugation affects the integrity of serum HDL. The nature of the chromatographically separated carriers of serum apoA-IV was investigated by quantitative immunoprecipitation. From these studies, it is concluded that apoA-IV in rat serum is present in at least three fractions: 1) particles with the size and composition of HDL, containing both apoA-I and apoA-IV and possibly minor quantities of apoE; 2) HDL-sized particles containing apoA-IV, but no apoA-I or apoE; 3) "free" apoA-IV probably containing small amounts of bound cholesterol and phospholipid.     

Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I

Endothelial lipase (EL) is a triglyceride lipase gene family member that has high phospholipase and low triglyceride lipase activity. The aim of this study was to determine whether the phospholipase activity of EL is sufficient to remodel HDLs into small particles and mediate the dissociation of apolipoprotein A-I (apoA-I). Spherical, reconstituted HDLs (rHDLs) containing apoA-I only [(A-I)rHDLs], apoA-II only [(A-II)rHDLs], or both apoA-I and apoA-II [(A-I/A-II) rHDLs] were prepared. The rHDLs, which contained only cholesteryl esters in their core and POPC on the surface, were incubated with EL. As the rHDLs did not contain triacylglycerol, only the POPC was hydrolyzed. Hydrolysis was greater in the (A-I/A-II)rHDLs than in the (A-I)rHDLs. The (A-II)rHDL phospholipids were not hydrolyzed by EL. EL remodeled the (A-I)rHDLs and (A-I/A-II)rHDLs, but not the (A-II)rHDLs, into smaller particles. The reduction in particle size was related to the amount of phospholipid hydrolysis, with the diameter of the (A-I/A-II)rHDLs decreasing more than that of the (A-I)rHDLs. These changes did not affect the conformation of apoA-I, and neither apoA-I nor apoA-II dissociated from the rHDLs. Comparable results were obtained when human plasma HDLs were incubated with EL. These results establish that the phospholipase activity of EL remodels plasma HDLs and rHDLs into smaller particles without mediating the dissociation of apolipoproteins.     

Distribution of apolipoprotein A-IV in human plasma

Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles.     

Interaction between high-density lipoprotein subpopulations in apo B-free and abetalipoproteinemic plasma.

Two populations of high-density lipoprotein (HDL) particles exist in human plasma. Both contain apolipoprotein (apo) A-I, but only one contains apo A-II: Lp(AI w AII) and Lp(AI w/o AII). To study the extent of interaction between these particles, apo B-free plasma prepared by the selective removal of apo B-containing lipoproteins (LpB) from the plasma of three normolipidemic (NL) subjects and whole plasma from two patients with abetalipoproteinemia (ABL) were incubated at 37 degrees C for 24 h. Apo B-free plasma samples were used to avoid lipid-exchange between HDL and LpB. Lp(AI w AII) and Lp(AI w/o AII) were isolated from each apo B-free plasma sample before and after incubation and their protein and lipid contents quantified. Before incubation, ABL plasma had reduced levels of Lp(AI w AII) and Lp(AI w/o AII), (40% and 70% of normals, respectively). Compared to the HDL of apo B-free NL plasma, ABL HDL had higher relative contents of free cholesterol, phospholipid and total lipid, and contained more particles with apparent hydrated Stokes diameter in the 9.2-17.0 nm region. These differences were particularly pronounced in particles without apo A-II. Despite their differences, the total cholesterol contents of Lp(AI w AII) increased, while that of Lp(AI w/o AII) decreased in all five plasma samples and the amount of apo A-I in Lp(AI w AII) increased by 6-8 mg/dl in four during the incubation. These compositional changes were accompanied by a relative reduction of particles in the 7.0-8.2 nm Stokes diameter size region and an increase of particles in the 9.2-11.2 nm region. These data are consistent with intravascular modulation between HDL particles with and without apo A-II. The observed increase in apo A-II-associated cholesterol and apo A-I, could involve either the transfer of cholesterol and apo A-I from particles without apo A-II to those with A-II, or the transfer of apo A-II from Lp(AI w AII) to Lp(AI w/o AII). The exact mechanism and direction of the transfer remain to be determined.