Isolation and partial characterization of lipoprotein A-II (LP-A-II) particles of human plasma.

High density lipoproteins (HDL) consist of a mixture of chemically and functionally distinct families of particles defined by their characteristic apolipoprotein (Apo) composition. The two major lipoprotein families are lipoprotein A-I (LP-A-I) and lipoprotein A-I:A-II (LP-A-I:A-II). This study describes the isolation of a third minor HDL family of particles referred to as lipoprotein A-II (LP-A-II) because it lacks ApoA-I and contains ApoA-II as its main or sole apolipoprotein constituent. Because ApoA-II is an integral protein constituent of three distinct lipoprotein families (LP-A-I:A-II, LP-A-II: B:C:D:E and LP-A-II), LP-A-II particles were isolated from whole plasma by sequential immunoaffinity chromatography on immunosorbers with antisera to ApoA-II, ApoB and ApoA-I, respectively. In normolipidemic subjects, the concentration of LP-A-II particles, based on ApoA-II content, is 4-18 mg/dl accounting for 5-20% of the total ApoA-II not associated with ApoB-containing lipoproteins. The lipid composition of LP-A-II particles is characterized by low percentage of triglycerides and cholesterol esters and a high percentage of phospholipids in comparison with lipid composition of LP-A-I and LP-A-II: A-II. The major part of LP-A-II particles contain ApoA-II as the sole apolipoprotein constituent; however, small subsets of LP-A-II particles may also contain ApoD and other minor apolipoproteins. The lipid/protein ratio of LP-A-II is higher than those of LP-A-I and LP-A-I:A-II. In homozygous ApoA-I and ApoA-I/ApoC-III deficiencies, LP-A-II particles are the only ApoA-containing high density lipoprotein with levels found to be within the same range (7-13 mg/dl) as those of normolipidemic subjects. However, in contrast to normal LP-A-II, their lipid composition is characterized by higher percentages of triglycerides and cholesterol esters and a lower percentage of phospholipids and their apolipoprotein composition by the presence of ApoC-peptides and ApoE in addition to ApoA-II and ApoD. These results show that LP-A-II particles are a minor HDL family and suggest that, in the absence of ApoA-I-containing lipoproteins, they become an efficient acceptor/donor of ApoC-peptides and ApoE required for a normal metabolism of triglyceride-rich lipoproteins. Their other possible functional roles in lipid transport remain to be established in future experiments.     

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.     

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)     

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.     

Characterization of lipoprotein produced by the perfused rhesus monkey liver

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.     

ApoA-II maintains HDL levels in part by inhibition of hepatic lipase. Studies In apoA-II and hepatic lipase double knockout mice.

High density lipoprotein (HDL) cholesterol levels are inversely related to the risk of developing coronary heart disease. Apolipoprotein (apo) A-II is the second most abundant HDL apolipoprotein and apoA-II knockout mice show a 70% reduction in HDL cholesterol levels. There is also evidence, using human apoA-II transgenic mice, that apoA-II can prevent hepatic lipase-mediated HDL triglyceride hydrolysis and reduction in HDL size. These observations suggest the hypothesis that apoA-II maintains HDL levels, at least in part, by inhibiting hepatic lipase. To evaluate this, apoA-II knockout mice were crossbred with hepatic lipase knockout mice. Compared to apoA-II-deficient mice, in double knockout mice there were increased HDL cholesterol levels (57% in males and 60% in females), increased HDL size, and decreased HDL cholesteryl ester fractional catabolic rate. In vitro incubation studies of plasma from apoA-II knockout mice, which contains largely apoA-I HDL particles, showed active lipolysis of HDL triglyceride, whereas similar studies of plasma from apoA-I knockout mice, which contains largely apoA-II particles, did not. In summary, these results strongly suggest that apoA-II is a physiological inhibitor of hepatic lipase and that this is at least part of the mechanism whereby apoA-II maintains HDL cholesterol levels.     

Role of apolipoprotein A-II in the structure and remodeling of human high-density lipoprotein (HDL): protein conformational ensemble on HDL

High-density lipoproteins (HDL, or "good cholesterol") are heterogeneous nanoparticles that remove excess cell cholesterol and protect against atherosclerosis. The cardioprotective action of HDL and its major protein, apolipoprotein A-I (apoA-I), is well-established, yet the function of the second major protein, apolipoprotein A-II (apoA-II), is less clear. In this review, we postulate an ensemble of apolipoprotein conformations on various HDL. This ensemble is based on the crystal structure of Δ(185-243)apoA-I determined by Mei and Atkinson combined with the "double-hairpin" conformation of apoA-II(dimer) proposed in the cross-linking studies by Silva's team, and is supported by the wide array of low-resolution structural, biophysical, and biochemical data obtained by many teams over decades. The proposed conformational ensemble helps integrate and improve several existing HDL models, including the "buckle-belt" conformation of apoA-I on the midsize disks and the "trefoil/tetrafoil" arrangement on spherical HDL. This ensemble prompts us to hypothesize that endogenous apoA-II (i) helps confer lipid surface curvature during conversion of nascent discoidal HDL(A-I) and HDL(A-II) containing either apoA-I or apoA-II to mature spherical HDL(A-I/A-II) containing both proteins, and (ii) hinders remodeling of HDL(A-I/A-II) by hindering the expansion of the apoA-I conformation. Also, we report that, although endogenous apoA-II circulates mainly on the midsize spherical HDL(A-I/A-II), exogenous apoA-II can bind to HDL of any size, thereby slightly increasing this size and stabilizing the HDL assembly. This suggests distinctly different effects of the endogenous and exogenous apoA-II on HDL. Taken together, the existing results and models prompt us to postulate a new structural and functional role of apoA-II on human HDL.     

Studies on the lipid and apolipoprotein compositions of two species of apoA-I-containing lipoproteins in normolipidemic males and females

Two species of apoA-I-containing lipoproteins (A-ILp), lipoprotein containing apoA-I and apoA-II (LpA-I/A-II) and lipoprotein containing apoA-I but no apoA-II (LpA-I), have been isolated from 20 normolipidemic adults (10 males and 10 females) by immunoaffinity chromatography. We have characterized the lipid and apolipoprotein compositions in these lipoproteins, and found sex differences. In A-ILp, the levels of lipids, except triglyceride, and the level of apoE were significantly higher in females than in males. In LpA-I/A-II, sex differences were found only in the levels of apoA-I and apoE. In LpA-I, the levels of all lipids, except triglyceride, and the level of apoA-I were significantly higher in females than in males. Therefore, sex differences observed in A-ILp appear to be due primarily to the differences found in LpA-I. Of considerable significance is our finding that the ratio of cholesteryl ester to total cholesterol in LpA-I was significantly lower than that in LpA-I/A-II in both males and females. This might suggest that LpA-I could be a carrier of free cholesterol.     

Relationship of the parameters of body cholesterol metabolism with plasma levels of HDL cholesterol and the major HDL apoproteins

The inverse relationship between plasma levels of high density lipoprotein (HDL) and coronary heart disease rates has suggested that HDL might influence body stores of cholesterol. Therefore, we have investigated potential relationships between the parameters of body cholesterol metabolism and the plasma levels of HDL cholesterol and the major HDL apoproteins. The study involved 55 human subjects who underwent long-term cholesterol turnover studies, as well as plasma lipoprotein and apolipoprotein assays. In order to maximize the likelihood of detecting existing relationships, the subjects were selected to span a wide range of plasma levels of lipids, lipoproteins, and apolipoproteins. Single univariate correlation analyses suggested weak but statistically significant inverse relationships of HDL cholesterol and apoA-I levels with the following model parameters: production rate (PR), the mass of rapidly exchanging body cholesterol (M1), the minimum estimate of the mass of slowly exchanging body cholesterol (M3min), and of the mass of total exchangeable body cholesterol (Mtotmin). These correlations, however, were quantitatively quite small (/r/ = 0.28-0.42) in comparison to the strength of the univariate relationships between body weight and PR (r = 0.76), M1 (r = 0.61), M3min (r = 0.58), and Mtotmin (r = 0.78). Correlations for apoA-II and apoE levels were even smaller than those for apoA-I and HDL cholesterol. In additional analyses using multivariate approaches, HDL cholesterol, apoA-I, apoA-II, and apoE levels were all found not to be independent determinants of the parameters of body cholesterol metabolism (/partial r/ less than 0.17, P greater than 0.3 in all cases). Thus the weak univariate correlations reflect relationships of HDL cholesterol and apoA-I levels with physiological variables, such as body size, which are primarily related to the model parameters. We conclude that plasma levels of HDL cholesterol and apoproteins A-I, A-II, and E are not quantitatively important independent determinants of the mass of slowly exchanging body cholesterol or of other parameters of long-term cholesterol turnover in humans. These studies give no support to the hypothesis that the inverse relationship between HDL cholesterol levels and coronary heart disease rates is mediated via an influence of HDL on body stores of cholesterol.     

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: the identification of lipoprotein B as the main lipoprotein degradation product

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.     

Speciated human high-density lipoprotein protein proximity profiles

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (～58%), phospholipids (～21%), total cholesterol (～16%), triglycerides (～5%), and free cholesterol (～4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.     

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.     

Differential tissue-specific expression of human apoA-I and apoA-II.

To evaluate the sources of high density lipoprotein (HDL) particles containing only apolipoprotein A-I (apoA-I), the synthesis of apoA-I and apolipoprotein A-II (apoA-II) was examined in human liver and small intestine as well as the human intestinally derived cell line, Caco-2. Human liver contained apoA-I, apoA-II as well as apolipoprotein B (apoB) mRNA. In contrast, human adult small intestine total and polyA+ RNA had little or no apoA-II despite the presence of apoA-I and apoB. Intestinal biopsies from normal individuals failed to show de novo apoA-II protein synthesis in the media of organ cultures during [35S]methionine pulse-chase labeling, whereas apoA-I could be readily detected. Caco-2 cells contained apoA-II mRNA and secreted apoA-II protein into the tissue culture media. These data indicate that the primary site of human apoA-II synthesis is in the liver and that the small intestine secretes apoA-I-containing high density lipoproteins.     

Lipid transfer particle-induced transformation of human high density lipoprotein into apolipoprotein A-I-deficient low density particles

Incubation of human high density lipoprotein (HDL) particles (density = 1.063-1.21 g/ml) with catalytic amounts of Manduca sexta lipid transfer particle (LTP) resulted in alteration of the density distribution of HDL protein such that the original HDL particles were transformed into new particles with an equilibrium density = 1.05 g/ml. Concomitantly, substantial amounts of protein were recovered in the bottom fraction of the density gradient. The LTP-induced alteration in HDL protein density distribution was dependent on the LTP concentration and incubation time. Electrophoretic analysis revealed that the lower density fraction contained apolipoprotein A-II (apoA-II) as the major apoprotein component while nearly all of the apoA-I was recovered in the bottom fraction. Lipid analysis of the HDL substrate and product fractions revealed that the apoA-I-rich fraction was nearly devoid of lipid (less than 1%, w/w). The lipid originally associated with HDL was recovered in the low density, apoA-II-rich, lipoprotein fraction, and the ratios of individual lipid classes were the same as in control HDL. Electron microscopy and gel permeation chromatography experiments revealed that the LTP-induced product lipoprotein population comprised particles of larger size (19.7 +/- 1.4-nm diameter) than control HDL (10.6 +/- 1.4-nm diameter). The results suggest that facilitated net lipid transfer between HDL particles altered the distribution of lipid such that apoprotein migration occurred and donor particles disintegrated. Similar results were obtained when human HDL3 or HDL2 density subclasses were employed as substrates for LTP. The lower surface area to core volume ratio of the larger, product lipoprotein particles compared with the substrate HDL requires that there be a decrease in the total exposed lipid/water interface which requires stabilization by apolipoprotein. Selective displacement of apoA-I by apoA-II or apoC, due to their greater surface binding affinity, dictates that apoA-I is preferentially lost from the lipoprotein surface and is therefore recovered as lipid-free apoprotein. Thus, it is conceivable that the structural arrangement of HDL particle lipid and apoprotein components isolated from human plasma may not represent the most thermodynamically stable arrangement of lipid and protein.     

Binding characteristics of high density lipoprotein subclasses to porcine liver, adrenal and skeletal muscle plasma membranes

1. We compared binding characteristics of 125I-labeled high density lipoprotein (HDL) subclasses to porcine liver, adrenal and skeletal muscle plasma membranes. 2. HDL subclasses were discriminated by their buoyant densities (HDL2 and HDL3) or by their apolipoprotein (apo) content (Lp-AI (particles containing apoA-I but no apoA-II) and LpA-I/A-II (particles containing both apoA-I and apoA-II)). 3. HDL2 and HDL3 showed saturable binding to the three types of membrane preparations. 4. No differences were found in the Kds within one HDL subclass. 5. Kds and maximal binding of HDL2 were lower than these of HDL3. Unlabeled HDL2 and HDL3, but not LDL, effectively displaced 125I-HDL2 and 125I-HDL3. 6. Binding of HDL was independent of the concentration of NaCl and did not require calcium. 7. These results suggest a process mediated by a single specific receptor in porcine liver, adrenal and skeletal muscle plasma membranes. 8. We also studied binding characteristics of HDL subclasses Lp-AI and LpA-I/A-II to porcine liver membranes. LpA-I showed the highest Kd and maximal binding. 9. All types of HDL subclasses studied (i.e. HDL2, HDL3, LpA-I and LpA-I/A-II) effectively competed for binding of both Lp-AI and LpA-I/A-II, suggesting that the HDL subclasses studied bind to the same receptor by their apoA-I moiety.     

Lipoprotein composition of human suction-blister interstitial fluid

Interstitial fluid (IF) was obtained in 27 apparently healthy subjects (12 males, 15 females) by applying mild suction (200-250 mm Hg) on the skin either on the midvolar forearm or on the paraumbilical region of the abdomen. The IF concentrations of lipids and apolipoproteins (apo) were studied and compared with those of serum (S). The mean ratio between interstitial fluid and serum (IF/S ratio) varied from 0.14 for forearm apoE to 0.29 for apoA-II on the abdomen. This ratio was consistently lower for apoE, C-II, C-III, and B than for apoA-I and A-II, and significantly lower on the arm than on the abdomen for all apolipoproteins studied. The IF/S ratios showed marked variations among individuals. However, interstitial fluid apolipoprotein concentrations at different blister sites were highly correlated within each individual. Studies with agarose gel electrophoresis and density gradient ultracentrifugation revealed that large triglyceride-rich particles were virtually lacking in interstitial fluid and that the relation between the low density lipoproteins (LDL) and high density lipoproteins (HDL) was shifted towards a greater proportion of HDL. The lipoprotein distribution in the HDL range of interstitial fluid differed from that of serum showing one maximum at a density of about 1.070 g/ml (serum HDL2 about 1.090 g/ml) and one at a density of 1.130-1.140 g/ml (serum HDL3, 1.110-1.120 g/ml). The former subfraction contained most of the lipoprotein-bound apoE while the latter contained the major part of apoA-I and apoA-II. Studies of the lipoproteins of interstitial fluid may add to our understanding of the development of atherosclerosis and xanthomatosis and may also provide valuable information on the permeability of the capillary membrane in normo- and pathophysiological states.     

Isolation and characterization of an apoA-II-containing lipoprotein (LP-A-II:B complex) from plasma very low density lipoproteins of patients with Tangier disease and type V hyperlipoproteinemia

Previous studies have shown that very low density lipoproteins (VLDL) from patients with Tangier disease are less effective as a substrate for human milk lipoprotein lipase (LPL) than VLDL from normal controls as assessed by measuring the first order rate constant (k1) of triglyceride hydrolysis. Tangier VLDL also has a higher content of apolipoprotein (apo) A-II than normal VLDL. To explore the possible relationship between the relatively high concentration of apoA-II in VLDL and low k1 values, Tangier VLDL were fractionated on an anti-apoA-II immunosorber. The retained fraction contained a newly identified triglyceride-rich lipoprotein characterized by the presence of apolipoproteins A-II, B, C-I, C-II, C-III, D, and E (LP-A-II:B:C:D:E or LP-A-II:B complex), whereas the unretained fraction consisted of previously identified triglyceride-rich apoB-containing lipoproteins free of apoA-II. In VLDL from patients with Tangier disease or type V hyperlipoproteinemia, the LP-A-II:B complex accounted for 70-90% and 25-70% of the total apoB content, respectively. The LP-A-II:B complexes had similar lipid and apolipoprotein composition; they were poor substrates for LPL as indicated by their low k1 values (0.014-0.016 min-1). In contrast, the apoA-II-free lipoproteins present in unretained fractions were effective substrates for LPL with k1 values equal to or greater than 0.0313 min-1. These results indicate that triglyceride-rich lipoproteins consist of several apoB-containing lipoproteins, including the LP-A-II:B complex, and that lipoprotein particles of similar size and density but distinct apolipoprotein composition also possess distinct metabolic properties.     

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.     

Apolipoprotein A-II modulates the binding and selective lipid uptake of reconstituted high density lipoprotein by scavenger receptor BI

High density lipoprotein (HDL) represents a mixture of particles containing either apoA-I and apoA-II (LpA-I/A-II) or apoA-I without apoA-II (LpA-I). Differences in the function and metabolism of LpA-I and LpA-I/A-II have been reported, and studies in transgenic mice have suggested that apoA-II is pro-atherogenic in contrast to anti-atherogenic apoA-I. The molecular basis for these observations is unclear. The scavenger receptor BI (SR-BI) is an HDL receptor that plays a key role in HDL metabolism. In this study we investigated the abilities of apoA-I and apoA-II to mediate SR-BI-specific binding and selective uptake of cholesterol ester using reconstituted HDLs (rHDLs) that were homogeneous in size and apolipoprotein content. Particles were labeled in the protein (with (125)I) and in the lipid (with [(3)H]cholesterol ether) components and SR-BI-specific events were analyzed in SR-BI-transfected Chinese hamster ovary cells. At 1 microg/ml apolipoprotein, SR-BI-mediated cell association of palmitoyloleoylphosphatidylcholine-containing AI-rHDL was significantly greater (3-fold) than that of AI/AII-rHDL, with a lower K(d) and a higher B(max) for AI-rHDL as compared with AI/AII-rHDL. Unexpectedly, selective cholesterol ester uptake from AI/AII-rHDL was not compromised compared with AI-rHDL, despite decreased binding. The efficiency of selective cholesterol ester uptake in terms of SR-BI-associated rHDL was 4-5-fold greater for AI/AII-rHDL than AI-rHDL. These results are consistent with a two-step mechanism in which SR-BI binds ligand and then mediates selective cholesterol ester uptake with an efficiency dependent on the composition of the ligand. ApoA-II decreases binding but increases selective uptake. These findings show that apoA-II can exert a significant influence on selective cholesterol ester uptake by SR-BI and may consequently influence the metabolism and function of HDL, as well as the pathway of reverse cholesterol transport.