Enhanced oxidative susceptibility and reduced antioxidant content of metabolic precursors of small, dense low-density lipoproteins.

PURPOSE: Elevated plasma concentrations of low-density lipoproteins (LDL) increase risk for coronary heart disease. However, lipoprotein profiles rich in small, dense LDL particles confer greater risk than those that mainly consist of large, buoyant LDL. This may be due, in part, to the greater oxidative susceptibility of small, dense LDL. In the current studies, we tested whether differences in the oxidative behavior of buoyant and dense LDL arise from differences in their immediate metabolic precursors, intermediate-density lipoproteins. SUBJECTS AND METHODS: We compared the properties of intermediate-density lipoproteins and buoyant and dense LDL subfractions in 9 subjects with the large, buoyant LDL phenotype versus 6 with the small, dense LDL phenotype. Oxidative susceptibility was evaluated based on conjugated diene formation and parinaric acid oxidation induced by copper. Antioxidants (ubiquinol-10 and alpha-tocopherol) were measured by high-performance liquid chromatography. RESULTS: Oxidative susceptibility was increased and antioxidant concentrations were decreased with increasing lipoprotein density (intermediate intermediate-density lipoproteins to buoyant LDL to dense LDL). Intermediate-density lipoproteins from subjects with the small, dense LDL phenotype had a greater oxidative susceptibility (by the parinaric acid test) and lower antioxidant concentrations than corresponding particles from subjects with the large, buoyant LDL phenotype. CONCLUSIONS: Differences in oxidative susceptibility between large, buoyant and small, dense LDL particles are apparent in their lipoprotein precursors. These results suggest that lipoprotein oxidative susceptibility may be metabolically programmed and that intermediate-density lipoproteins may contribute to the increased risk associated with the small, dense LDL phenotype.

Overexpression of human hepatic lipase and ApoE in transgenic rabbits attenuates response to dietary cholesterol and alters lipoprotein subclass distributions.

The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism.

Influence of apoE content on receptor binding of large, bouyant LDL in subjects with different LDL subclass phenotypes.

We investigated the influence of apolipoprotein (apo) E-containing particles on LDL receptor binding of large, buoyant LDL subfractions (LDL I) from subjects with predominantly large (phenotype A) and small (phenotype B) LDL particles. Direct binding by human fibroblast LDL receptors was tested at 4 degrees C before and after removal of apoE-containing particles by immunoaffinity chromatography. The binding affinity of total LDL I in phenotype B was greater than that in phenotype A (Kd of 1.83+/-0.3 and 3.43+/-0.9 nmol/L, respectively, P<.05). LDL I from phenotype B subjects had a higher apoE to apoB molar ratio than did that from phenotype A (0.16+/-0.04 versus 0.06+/-0.02, P<.05). Nondenaturing gradient gel electrophoresis of apoE-containing LDL I isolated by immunoaffinity chromatography revealed a substantially larger peak particle diameter than in apoE-free LDL I, and comparison of LDL I composition before and after immunoaffinity chromatography suggested an increase in triglyceride content of apoE-containing particles. After removal of these particles, there was a greater than twofold reduction in LDL receptor affinity of phenotype B LDL (Kd of 1.83+/-0.3 to 3.76+/-0.6, P<.01), whereas in phenotype A no change was observed (Kd of 3.43+/-0.9 to 3.57+/-0.4, respectively). The receptor affinity of apoE-free LDL I from phenotype A and B subjects did not differ. These findings confirm that large, buoyant LDL particles from phenotype B subjects have a higher LDL receptor affinity than does LDL I from phenotype A subjects and suggest that this difference is due to an increased content of large, triglyceride-enriched, apoE-containing lipoproteins. It is possible that the accumulation of these particles reflects abnormalities in the metabolism of remnant lipoproteins that contribute to atherosclerosis risk in phenotype B subjects.

Variability of plasma HDL subclass concentrations in men and women over time.

Plasma HDL subclasses were examined by gradient gel electrophoresis in repeated samples to assess variability over time. Absorbance of the protein stain was used as an index of mass concentrations at 0.01-nm intervals within five HDL subclasses: HDL3c (7.2 to 7.8 nm), HDL3b (7.8 to 8.2 nm), HDL3a (8.2 to 8.8 nm), HDL2a (8.8 to 9.7 nm), and HDL2b (9.7 to 12 nm). Three separate longitudinal studies of men showed that repeated samples of HDL over time were correlated most strongly within HDL2b, somewhat less within HDL2a, and more weakly within HDL3a, HDL3b, and HDL3c. As in men, repeated samples in women from two studies were significantly correlated within the HDL2b, HDL2a, and HDL3b intervals. Plasma HDL2b levels were significantly more stable in men than in women. Although the variability of HDL subclass measurements includes both methodological and physiological sources, differences in laboratory measurement error do not appear to explain the differences in correlations among subclasses. Specifically, analysis of 288 replications from frozen aliquots suggested that laboratory error had the least effect on correlations involving HDL3 subclasses and only slightly greater effect on correlations involving HDL2 subclasses. Our results suggest that for plasma sampled over time, the stability of HDL subclass levels increases with particle size. Prior reports of subclass-specific correlations between HDL and other variables (eg, diet, exercise, and other lipids) are unlikely to be artifacts of laboratory precision but could arise from subclass differences in variability that are physiological.

Charge properties of low density lipoprotein subclasses.

Measurements of electrophoretic mobility and particle size of low density lipoproteins (LDL) allowed use of standard electrokinetic theory to quantitate LDL charge characteristics from subjects with predominance of large LDL (pattern A, n = 9) or small LDL (pattern B, n = 8). Pattern A LDL was found to have significantly lower (P < or = 0.001) mobility (-0.22 +/- 0.01 micron s-1 cm V-1), surface potential (-4.2 +/- 0.3 mV) and charge density (-500 +/- 34 esu/cm2) than pattern B LDL (-0.25 +/- 0.01 micron s-1 cm V-1, -4.9 +/- 0.3 mV, and -580 +/- 30 esu/cm2), but no significant difference in particle valence (-22.0 +/- 1.4 for pattern A vs. -21.8 +/- 1.9 for pattern B). Thus, the greater mobility of pattern B LDL is due to similar net charge residing on a smaller particle. Comparison of subfractions in pattern B relative to pattern A LDL revealed greater surface potential in all pattern B subfractions and greater charge density in fractions of d > or = 1.032 g/ml. In a subset of subjects incubation with neuraminidase produced significant reductions in all LDL charge parameters for all subfractions, but did not abolish the differences between pattern A and B. Thus increased surface potential and charge density of unfractionated pattern B LDL is due both to charge properties of particles across the size and density spectrum as well as enrichment of pattern B LDL with smaller, denser particles that have higher surface charge density.

A prospective study of triglyceride level, low-density lipoprotein particle diameter, and risk of myocardial infarction.

OBJECTIVE: To test whether a predominance of small, dense low-density lipoprotein (LDL) particles and elevated triglyceride levels are independent risk factors for myocardial infarction (MI). DESIGN: Nested case-control study with prospectively collected samples. SETTING: Prospective cohort study. PARTICIPANTS: Blood samples were collected at baseline (85% nonfasting samples) from 14916 men aged 40 to 84 years in the Physicians' Health Study. MAIN OUTCOME MEASUREMENTS: Myocardial infarction diagnosed during 7 years of follow-up. RESULTS: Cases (n=266) had a significantly smaller LDL diameter (mean [SD], 25.6 [0.9] nm) than did controls (n=308) matched on age and smoking (mean [SD], 25.9 [8] nm; P<.001). Cases also had higher median triglyceride levels (1.90 vs 1.49 mmol/L [168 vs 132 mg/dL]; P<.001). The LDL diameter had a high inverse correlation with triglyceride level (r=-0.71), and a high direct correlation with high-density lipoprotein cholesterol (HDL-C) level (r=0.60). We observed a significant multiplicative interaction between triglyceride and total cholesterol (TC) levels (P=.01). After simultaneous adjustment for lipids and a variety of coronary risk factors, LDL particle diameter was no longer a statistically significant risk indicator, with a relative risk (RR) of 1.09 (95% confidence interval [CI], 0.85-1.40) per 0.8-nm decrease. However, triglyceride level remained significant with an RR of 1.40 (95% CI, 1.10-1.77) per 1.13 mmol/L (100-mg/dL) increase. The association between triglyceride level and MI risk appeared linear across the distribution; men in the highest quintile had a risk about 2.5 times that of those in the lowest quintile. The TC level, but not HDL-C level, also remained significant, with an RR of 1.80 (95% CI, 1.44-2.26) per 1.03-mmol/L (40-mg/dL) increase. CONCLUSIONS: These findings indicate that nonfasting triglyceride levels appear to be a strong and independent predictor of future risk of MI, particularly when the total cholesterol level is also elevated. In contrast, LDL particle diameter is associated with risk of MI, but not after adjustment for triglyceride level. Increased triglyceride level, small LDL particle diameter, and decreased HDL-C levels appear to reflect underlying metabolic perturbations with adverse consequences for risk of MI; elevated triglyceride levels may help identify high-risk individuals.

Multilocus genetic determinants of LDL particle size in coronary artery disease families.

Recent interest in atherosclerosis has focused on the genetic determinants of low-density lipoprotein (LDL) particle size, because of (i) the association of small dense LDL particles with a three-fold increased risk for coronary artery disease (CAD) and (ii) the recent report of linkage of the trait to the LDL receptor (chromosome 19). By utilizing nonparametric quantitative sib-pair and relative-pair analysis methods in CAD families, we tested for linkage of a gene or genes controlling LDL particle sizes with the genetic loci for the major apolipoproteins and enzymes participating in lipoprotein metabolism. We confirmed evidence for linkage to the LDL receptor locus (P=.008). For six candidate gene loci, including apolipoprotein(apo)B, apoAII, apo(a), apoE-CI-CII, lipoprotein lipase, and high-density lipoprotein-binding protein, no evidence for linkage was observed by sib-pair linkage analyses (P values ranged from .24 to .81). However, in addition, we did find tentative evidence for linkage with the apoAI-CIII-AIV locus (chromosome 11) (P=.06) and significant evidence for linkage of the cholesteryl ester transfer protein locus (chromosome 16) (P=.01) and the manganese superoxide dismutase locus (chromosome 6) (P=.001), thus indicating multilocus determination of this atherogenic trait.

Transformation of HepG2 nascent lipoproteins by LCAT: modulation by HepG2 d > 1.235 g/ml fraction.

We have previously shown that lecithin:cholesterol acyltransferase (LCAT) can transform ultracentrifugally isolated HepG2 lipoproteins (d < 1.235 g/ml) into particles that differ substantially from their nascent precursors. Transformed high density lipoprotein (HDL) subpopulations, as judged by nondenaturing gradient gel electrophoresis (GGE), resemble plasma HDL, i.e., HDL2a- and HDL3a-sized particles predominate. In HepG2 conditioned medium (CM), 60-70% of apoA-I is in the d > 1.235 g/ml fraction (lipid-poor apoA-I); hence we investigated whether inclusion of d > 1.235 g/ml fraction in LCAT incubations altered HDL subpopulations. After 18 h incubation of CM (containing lipoproteins and d > 1.235 g/ml fraction) with purified LCAT, the major transformation product on GGE was a large 9.7-nm particle (HDL2b pattern); a minor component appeared at 7.4 nm (HDL3c). Differences in particle size distribution between CM and isolated lipoprotein incubations were not the result of differences in LCAT activity; mass ratios of unesterified cholesterol:cholesteryl ester and phospholipid:cholesteryl ester were similar. Removal of apoA-I from the d > 1.235 g/ml fraction by immunoaffinity chromatography prior to incubation with the d < 1.235 g/ml fraction produced the same products (i.e., HDL2b pattern) as incubations performed with the unaltered d > 1.235 g/ml fraction; therefore, lipid-poor apoA-I does not influence nascent HDL transformation. Cholesteryl ester was transferred from HepG2 HDL to LDL in CM incubations; however, cholesteryl ester transfer protein was not immunochemically identified. Removal of HepG2 LDL from CM prior to incubation with LCAT still resulted in the HDL2b pattern. We conclude that HepG2 cells secrete a factor(s) that modifies nascent HDL transformation products into a predominantly HDL2b subpopulation.

Lipoprotein subclasses in genetic studies: the Berkeley data set.

In conjunction with a study examining the inheritance of LDL subclass patterns in a healthy population, measurements of lipids, lipoproteins, and lipoprotein subclasses were performed in 301 individuals in 27 kindreds. Questionnaires were used to obtain information on use of medications, hormones, cigarettes, and alcohol. Laboratory data from this study (the Berkeley data set) include measurements of LDL and HDL size subclasses by nondenaturing gradient gel electrophoresis, and measurement of apolipoprotein A-I by radial immunodiffusion.

Production of polyacrylamide gradient gels for the electrophoretic resolution of lipoproteins.

We describe a protocol to cast nondenaturing polyacrylamide gradient gels (SFBR3/31) for the size resolution of lipoproteins. The protocol yields gels with minimal lot-to-lot variation in length and electrophoretic properties. Absorbance profiles of cholesterol-stained lipoproteins in baboon sera were used to estimate the relative amounts of stain in four lipoprotein size classes (VLDL+LDL, HDL1, HDL2, and HDL3). When compared with gels from a commercial source, the SFBR3/31 gels gave very similar results in terms of precision (coefficients of variation) and of estimated amounts of lipoproteins in the four size classes. In other studies, we estimated peak diameters of protein-stained human lipoproteins after calibrating the gels with size standards. Peak diameters estimated using SFBR3/31 gels were highly correlated (r2 = 0.99, n = 33) with those estimated using gels from a commercial source. We conclude that the protocol reliably produces gradient gels that are suitable for the analysis of lipoprotein phenotypes.

Apolipoprotein-specific populations in high density lipoproteins of human cord blood.

High density lipoproteins (HDL) in human cord blood have previously been shown to exhibit particle size profiles distinctly different from those of adult HDL. The adult HDL profile is comprised of separate contributions from two major apolipoprotein-specific populations; one population contains both apolipoproteins AI and AII (HDL(AIwAII], while the other has apolipoprotein AI without AII (HDL(AIw/oAII]. The present studies establish that cord blood HDL are also comprised of HDL(AIwAII) and HDL(AIw/oAII) populations whose particle size profiles closely reflect cholesterol and HDL-cholesterol levels in cord blood. Compared with the adult, cord blood HDL(AIwAII) profiles generally show both a greater subspeciation within HDL2a and HDL3b/3c size intervals as well as relative reduction of material in the HDL3a interval. In the cord blood HDL(AIw/oAII) profile, HDL2b(AIw/oAII) particles also show subspeciation with a major component that is consistently larger than that normally observed in the adult (11.2 vs. 10.3 nm). As in the adult, the HDL3a(AIw/oAII) component is present but, unlike the adult, its relative amount is low; hence, its peak is usually not discernable in the cord blood total HDL profile. Our studies show that the larger-sized HDL2b(AIw/oAII) of cord blood are enriched in phospholipid which probably accounts for their increased size. The protein moiety of the larger-sized HDL2b(AIw/oAII) has a molecular weight equivalent to four apolipoprotein AI molecules per particle similar to the normal-sized adult subpopulation. Phospholipid enrichment of cord blood HDL(AIwAII) subpopulations within the HDL2a size interval was not observed. However, the protein moiety of cord blood HDL2a(AIwAII) is unusual in that it exhibits an apolipoprotein AI:AII molar ratio considerably lower (0.8:1 vs. 1.6:1) than that of adult. We suggest that the unique particle size distribution of cord blood total HDL is due in large part to: (a) a specific enrichment of phospholipid in HDL2b(AIw/oAII) species, producing particles larger than normal adult counterparts and (b) an elevated proportion of apoAII carried by the HDL(AIwAII) particles that may influence subspeciation in the HDL3a/b/c size interval.

Dissociation of high density lipoprotein precursors from apolipoprotein B-containing lipoproteins in the presence of unesterified fatty acids and a source of apolipoprotein A-I.

Incubation of low (LDL), intermediate (IDL), or very low density lipoproteins (VLDL) with palmitic acid and either high density lipoproteins (HDL), delipidated HDL, or purified apolipoprotein (apo) A-I resulted in the formation of lipoprotein particles with discoidal structure and mean particle diameters ranging from 146 to 254 A by electron microscopy. Discs produced from IDL or LDL averaged 26% protein, 42% phospholipid, 5% cholesteryl esters, 24% free cholesterol, and 3% triglycerides; preparations derived from VLDL contained up to 21% triglycerides. ApoA-I was the predominant protein present, with smaller amounts of apoA-II. Crosslinking studies of discs derived from LDL or IDL indicated the presence of four apoA-I molecules per particle, while those derived from large VLDL varied more in size and contained as many as six apoA-I molecules per particle. Incubation of discs derived from IDL or LDL with purified lecithin:cholesterol acyltransferase (LCAT), albumin, and a source of free cholesterol produced core-containing particles with size and composition similar to HDL2b. VLDL-derived discs behaved similarly, although the HDL products were somewhat larger and more variable in size. When discs were incubated with plasma d greater than 1.21 g/ml fraction rather than LCAT, core-containing particles in the size range of normal HDL2a and HDL3a were also produced. A variety of other purified free fatty acids were shown to promote disc formation. In addition, some mono and polyunsaturated fatty acids facilitated the formation of smaller, spherical particles in the size range of HDL3c. Both discoidal and small spherical apoA-I-containing lipoproteins were generated when native VLDL was incubated with lipoprotein lipase in the presence of delipidated HDL. We conclude that lipolysis product-mediated dissociation of lipid-apoA-I complexes from VLDL, IDL, or LDL may be a mechanism for formation of HDL subclasses during lipolysis, and that the availability of different lipids may influence the type of HDL-precursors formed by this mechanism.

Effects of atherogenic diet consumption on lipoproteins in mouse strains C57BL/6 and C3H.

Inbred mouse strains C57BL/6J (B6) (susceptible) and C3H/HeJ (C3H) (resistant) differ in atherosclerosis susceptibility due to a single gene, Ath-1. Plasma lipoproteins from female mice fed chow or an atherogenic diet displayed strain differences in lipoprotein particle sizes and apolipoprotein (apo) composition. High density lipoprotein (HDL) particle sizes were 9.5 +/- 0.1 nm for B6 and 10.2 +/- 0.1 nm for C3H. No major HDL particle size subclasses were observed. Plasma HDL level in the B6 strain was reduced by the atherogenic diet consumption while the HDL level in the resistant C3H mice was unaffected. The reduction in HDL in the B6 strain was associated with decreases in HDL apolipoproteins A-I(-34%) and A-II(-60%). The HDL apoC content in mice fed chow was two-fold higher in C3H than B6. Lipoproteins containing apolipoprotein B (VLDL, IDL, LDL) shifted from a preponderance of the B-100 (chow diet) to a preponderance of the B-48 (atherogenic diet). The LDL-particle size distribution was strain-specific with the chow diet but not genetically associated with the Ath-1 gene. In both strains on each diet, apolipoprotein E was largely distributed in the VLDL, LDL, and HDL fractions. The B6 strain became sixfold elevated in total lipoprotein E content which in the C3H strain was not significantly affected by diet. However, the C3H LDL apoE content was reduced. On both diets, the C3H strain exhibited apolipoprotein E levels comparable to the atherogenic diet-induced levels of the B6 mice.

Discoidal complexes containing apolipoprotein E and their transformation by lecithin-cholesterol acyltransferase.

The primary objectives of this study were to determine whether analogs to native discoidal apolipoprotein (apo)E-containing high-density lipoproteins (HDL) could be prepared in vitro, and if so, whether their conversion by lecithin-cholesterol acyltransferase (LCAT; EC 2.3.1.43) produced particles with properties comparable to those of core-containing, spherical, apoE-containing HDL in human plasma. Complexes composed of apoE and POPC, without and with incorporated unesterified cholesterol, were prepared by the cholate-dialysis technique. Gradient gel electrophoresis showed that these preparations contain discrete species both within (14-40 nm) and outside (10.8-14 nm) the size range of discoidal apoE-containing HDL reported in LCAT deficiency. The isolated complexes were discoidal particles whose size directly correlated with their POPC:apoE molar ratio: increasing this ratio resulted in an increase in larger complexes and a reduction in smaller ones. At all POPC:apoE molar ratios, size profiles included a major peak corresponding to a discoidal complex 14.4 nm long. Preparations with POPC:apoE molar ratios greater than 150:1 contained two distinct groups of complexes, also in the size range of discoidal apoE-containing HDL from patients with LCAT deficiency. Incorporation of unesterified cholesterol into preparations (molar ratio of 0.5:1, unesterified cholesterol:POPC) resulted in component profiles exhibiting a major peak corresponding to a discoidal complex 10.9 nm long. An increase of unesterified cholesterol and POPC (at the 0.5:1 molar ratio) in the initial mixture, increased the proportion of larger complexes in the profile. Incubation of isolated POPC-apoE discoidal complexes (mean sizes, 14.4 and 23.9 nm) with purified LCAT and a source of unesterified cholesterol converted the complexes to spherical, cholesteryl ester-containing products with mean diameters of 11.1 nm and 14.0 nm, corresponding to apoE-containing HDL found in normal plasma. Conversion of smaller cholesterol-containing discoidal complexes (mean size, 10.9 nm) under identical conditions resulted in spherical products 11.3, 13.3, and 14.7 nm across. The mean sizes of these conversion products compared favorably with those (mean diameter, 12.3 nm) of apoE-containing HDL of human plasma. This conversion of cholesterol-containing complexes is accompanied by a shift of some apoE to the LDL particle size interval. Our study indicates that apoE-containing complexes formed by the cholate-dialysis method include species similar to discoidal apoE-containing HDL and that incubation with LCAT converts most of them to spherical core-containing particles in the size range of plasma apoE-containing HDL. Plasma HDL particles containing apoE may arise in part from direct conversion of discoidal apoE-containing HDL by LCAT.

Lecithin:cholesterol acyltransferase-induced transformation of HepG2 lipoproteins.

Previous studies with the human hepatoblastoma-derived HepG2 cell line in this laboratory have shown that these cells produce high density lipoproteins (HDL) that are similar to HDL isolated from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Experiments were, therefore, performed to determine whether HepG2 HDL could be transformed into plasma-like particles by incubation with LCAT. Concentrated HepG2 lipoproteins (d less than 1.235 g/ml) were incubated with purified LCAT or lipoprotein-deficient plasma (LPDP) for 4, 12, or 24 h at 37 degrees C. HDL isolated from control samples possessed excess phospholipid and unesterified cholesterol relative to plasma HDL and appeared as a mixed population of small spherical (7.8 +/- 1.3 nm) and larger discoidal particles (17.7 +/- 4.9 nm long axis) by electron microscopy. Nondenaturing gradient gel analysis (GGE) of control HDL showed major peaks banding at 7.4, 10.0, 11.1, 12.2, and 14.7 nm. Following 4-h LCAT and 12-h LPDP incubations, HepG2 HDL were mostly spherical by electron microscopy and showed major peaks at 10.1 and 8.1 nm (LCAT) and 10.0 and 8.4 nm (LPDP) by GGE; the particle size distribution was similar to that of plasma HDL. In addition, the chemical composition of HepG2 HDL at these incubation times approximated that of plasma HDL. Molar increases in HDL cholesteryl ester were accompanied by equimolar decreases in phospholipid and unesterified cholesterol. HepG2 low density lipoproteins (LDL) isolated from control samples showed a prominent protein band at 25.6 nm with GGE. Active LPDP or LCAT incubations resulted in the appearance of additional protein bands at 24.6 and 24.1 nm. No morphological changes were observed with electron microscopy. Chemical analysis indicated that the LDL cholesteryl ester formed was insufficient to account for phospholipid lost, suggesting that LCAT phospholipase activity occurred without concomitant cholesterol esterification.

Conversion of apolipoprotein-specific high-density lipoprotein populations during incubation of human plasma.

Incubation studies were performed on plasma obtained from subjects selected for relatively low levels of high-density lipoprotein cholesterol (HDL-C) (no greater than 30 mg/dl) and particle size distributions enriched in the HDL3 subclass. Incubation (12 h, 37 degrees C) of plasma in the presence or absence of lecithin: cholesterol acyltransferase activity produces marked alteration in size profiles of both major apolipoprotein-specific HDL3 populations (HDL3(AI w AII), HDL3 species containing both apolipoprotein A-I and apolipoprotein A-II, and HDL3(AI w/o AII), HDL3 species containing apolipoprotein A-I) as isolated by immunoaffinity chromatography. In the presence or absence of lecithin: cholesterol acyltransferase activity, plasma incubation results in a shift of HDL3(AI w AII) species (initial mean sizes of major components, approx. 8.8 and 8.0 nm) predominantly to larger particles (mean size, 9.8 nm). A less prominent shift to smaller particles (mean size, 7.8 nm) accompanies the conversion to larger particles only when the enzyme is active. Combined shifts to larger (mean size, 9.8 nm) and smaller (mean size, 7.4 nm) particles are observed for HDL3(AI w/o AII) particles (mean size, 8.3 nm) also only in the presence of enzyme activity. However, in the absence of enzyme activity, HDL3(AI w/o AII) species, unlike the HDL3(AI w AII) species, are converted to smaller (mean size 7.4 nm) rather than to larger particles. Like native HDL2b(AI w/o AII) particles, the larger HDL3(AI w/o AII) conversion products exhibit a protein moiety with molecular weight equivalent to four apolipoprotein A-I molecules per particle; small HDL3(AI w/o AII) products are comprised predominantly of particles with two apolipoprotein A-I per particle. Incubation-induced conversion of HDL3 particles in the presence of lecithin: cholesterol acyltransferase activity is associated with increased binding of both apolipoprotein-specific HDL populations to low-density lipoproteins (LDL). The present studies indicate that, in the absence of lecithin: cholesterol acyltransferase activity, the two HDL3 populations follow different conversion pathways, possibly due to apolipoprotein-specific activities of lipid transfer protein or conversion protein in plasma. Our studies also suggest that lecithin: cholesterol acyltransferase activity may play a role in the origins of large HDL2b(AI w/o AII) species in human plasma by participating in the conversion of HDL3(AI w/o AII) particles, initially with three apolipoprotein A-I, to larger particles with four apolipoprotein A-I per particle.

Transformation of large discoidal complexes of apolipoprotein A-I and phosphatidylcholine by lecithin-cholesterol acyltransferase.

Using a cholate-dialysis recombination procedure, complexes of apolipoprotein A-I and synthetic phosphatidylcholine (1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC] were prepared in mixtures at a relatively high molar ratio of 150:1 phosphatidylcholine/apolipoprotein A-I. Particle size distribution analysis by gradient gel electrophoresis of the recombinant mixtures indicated the presence of a series of discrete complexes that included species migrating at RF values observed for discoidal particles in nascent high-density lipoproteins (HDL) in plasma of lecithin-cholesterol acyltransferase-deficient subjects. One of these complex species, designated complex class 6, formed with either phosphatidylcholine, was isolated by gel filtration and characterized at follows: discoidal shape (mean diameter 20.8 nm (POPC) and 19.0 nm (DOPC]; molar ratio, phosphatidylcholine/apolipoprotein A-I, 155:1 (POPC) and 130:1 (DOPC); and both containing 4 molecules of apolipoprotein A-I per particle. Incubation of class 6 complexes with lecithin-cholesterol acyltransferase (EC 2.3.1.43) and a source of unesterified cholesterol (low-density lipoprotein (LDL] was shown by electron microscopy to result in a progressive transformation of the discoidal particles (0 h) to deformable (2.5 h) and to spherical particles (24 h). The spherical particles (diameter 13.6 nm (POPC) and 12.5 nm (DOPC) exhibit sizes at the upper boundary of the interval defining the human plasma (HDL2b)gge (12.9-9.8 nm). The spherical particles contain a cholesteryl ester core that reaches a limiting molar ratio of approx. 50-55:1 cholesteryl ester/apolipoprotein A-I. The deformable particles assume a rectangular shape under negative staining and, relative to the 24-h spherical product, are enriched in phosphatidylcholine. Chemical crosslinking (by dimethyl suberimidate) of the isolated transformation products shows the 24-h spherical particle to contain predominantly 4 apolipoprotein A-I molecules; products produced after intermediate periods of time appear to contain species with 3 and 4 apolipoproteins per particle. Our in vitro studies indicate a potential pathway in the origins of large, apolipoprotein A-I-containing plasma HDL particles. The deformable species observed during transformation were similar in size and shape to particles observed in interstitial fluid.

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 complexes of egg yolk phosphatidylcholine and apolipoprotein A-II prepared in the absence and presence of sodium cholate.

Complexes of apolipoprotein A-II and egg yolk phosphatidylcholine were prepared in mixtures of different composition in the absence and presence of sodium cholate. By gradient gel electrophoresis, complex preparations were polydisperse and particle size distributions were influenced by the composition of the reconstitution mixture. Complexes generally exhibited a discoidal morphology by electron microscopy, but showed increased formation of vesicular complexes at elevated levels of egg yolk PC in the mixtures. By chemical crosslinking, complexes formed in the absence of cholate were shown to consist primarily of discoidal species with three apolipoprotein A-II molecules per particle in the mixtures investigated; complexes formed in the presence of cholate included species ranging from three to five apolipoprotein A-II per particle. The number of apolipoprotein A-II per particle and the sizes of the complexes, prepared in cholate, increased with increase of egg yolk PC in the reconstitution mixture. Relative to the particle size distribution of discoidal complexes formed in the absence of cholate, those prepared in cholate showed a distribution shifted to larger particle sizes. Complexes of similar particle size distribution formed in the presence or absence of cholate showed similar physical-chemical properties. Discoidal complexes with the same number of apolipoprotein A-II per particle but of different size and composition were observed, suggesting the possibility of some conformational adaptation of apolipoprotein A-II leading to stabilization of egg yolk PC bilayers of different diameter. Properties of particle size distributions of discoidal complexes prepared in cholate of apolipoprotein A-II and egg yolk PC were compared with those of complexes of apolipoprotein A-I previously reported (Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) Biochim. Biophys. Acta 750, 353-364).

Pathways in the formation of human plasma high density lipoprotein subpopulations containing apolipoprotein A-I without apolipoprotein A-II.

The lecithin:cholesterol acyltransferase (LCAT)-induced transformation of two discrete species of model complexes that differ in number of apolipoprotein A-I (apoA-I) molecules per particle was investigated. One complex species (designated 3A-I(UC)-complexes) contained 3 apoA-I per particle, was discoidal (13.5 X 4.4 nm), and had a molar composition of 22:78:1 (unesterified cholesterol (UC):egg yolk phosphatidylcholine (egg yolk PC):apoA-I). The other complex species (designated 2A-I(UC)complexes) containing 2 apoA-I per particle was also discoidal (8.4 X 4.1 nm) and had a molar composition of 6:40:1. Transformation of 3A-I(UC)complexes by partially purified LCAT yielded a product (24 hr, 37 degrees C) with a cholesteryl ester (CE) core, 3 apoA-I, and a mean diameter of 9.2 nm. The 2A-I(UC)complexes were only partially transformed to a core-containing product (24 hr, 37 degrees C) which also had 3 apoA-I; this product, however, was smaller (diameter of 8.5 nm) than the product from 3A-I(UC)complexes. Transformation of 3A-I(UC)complexes appeared to result from build-up of core CE directly within the precursor complex. Transformation of 2A-I(UC)complexes, however, followed a stepwise pathway to the product with 3 apoA-I, apparently involving fusion of transforming precursors and release of one apoA-I from the fusion product. In the presence of low density lipoprotein (LDL), used as a source of additional cholesterol, conversion of 2A-I(UC)complexes to the product with 3 apoA-I was more extensive. The transformation product of 3A-I(UC)complexes in the presence of LDL also had 3 apoA-I but was considerably smaller in size (8.6 vs. 9.2 nm, diameter) and had a twofold lower molar content of PC compared with the product formed without LDL. LDL appeared to act both as a donor of UC and an acceptor of PC. Transformation products with 3 apoA-I obtained under the various experimental conditions in the present studies appear to be constrained in core CE content (between 13 to 22 CE per apoA-I; range of 9 CE molecules) but relatively flexible in content of surface PC molecules they can accommodate (between 24 to 49 PC per apoA-I; range of 25 PC molecules). The properties of the core-containing products with 3 apoA-I compare closely with those of the major subpopulation of human plasma HDL in the size range of 8.2-8.8 nm that contains the molecular weight equivalent of 3 apoA-I molecules.