HDL antioxidant effects as assessed using a nonexchangeable probe to monitor particle-specific peroxidative stress in LDL-HDL mixtures.

High density lipoproteins (HDL) have been reported to inhibit oxidation of low density lipoproteins (LDL) based in part on observations that oxidative changes occur more slowly in LDL-HDL mixtures than in LDL alone. In the current studies, we developed an approach to discern particle-specific oxidation kinetics within mixed particle systems using the oxidation-labile fluorescent probe parinaric acid cholesteryl ester (PnCE) and applied this to the study of HDL inhibition effects. PnCE was introduced into acceptor lipoproteins by cholesteryl ester transfer protein (CETP)-mediated transfer from donor microemulsions. Incubation of PnCE-containing LDL and HDL with non-probe-containing HDL and LDL, respectively, followed by measurement of reisolated fractions, indicated that PnCE does not transfer appreciably between lipoprotein fractions. Oxidative loss of lipoprotein-associated PnCE occurred essentially in tandem with changes in conjugated dienes, suggesting that PnCE loss reflects the course of peroxidation of endogenous lipoprotein lipids. Using PnCE to separately monitor LDL- and HDL-specific oxidation within LDL-HDL mixtures, we obtained direct evidence that HDL inhibits both Cu(2+)- and Fe(3+)-induced peroxidation of LDL-associated lipids. Notably, in the presence of Cu2+, loss of HDL-associated PnCE fluorescence also was inhibited in LDL-HDL co-incubations, suggesting that LDL exert an antioxidant effect under these conditions as well. Thus, results obtained using this new methodology are consistent with previously reported antioxidant effects of HDL, but indicate that the behavior of individual lipoprotein particles may be more complicated than can be predicted from the collective behavior of the lipoprotein mixture.

Human apo A-I in transgenic mice is more efficient in activating lecithin:cholesterol acyltransferase than mouse apo A-I.

This study shows that, in control and transgenic mice, there is a parallel increase in LCAT activity and plasma apo A-I concentrations during postnatal development. We also demonstrate that human apo A-I is a much more efficient activator (1.6-fold) of mouse LCAT activity than mouse apo A-I. We propose that the differences in amino acid sequence between human and mouse apo A-I may account for the higher LCAT activity with human apo A-I.

Sodium oleate-facilitated reassembly of apolipoprotein A-I with phosphatidylcholine.

The influence of sodium oleate (oleate) on complexing of apolipoprotein A-I (apo A-I) with egg yolk phosphatidylcholine (EYPC) was evaluated. Without the use of additional detergent such as sodium cholate, oleate facilitates formation of a single complex of unique stoichiometry, approx. 76:2:20, molar ratio EYPC/apo A-I/oleate, and mean size 7.4 nm with round to ellipsoidal morphology. Near complete reassembly of apo A-I into the complex occurs when the stoichiometry of the mixture approximates that of the complex itself. With increasing content of EYPC in the mixture, the same complex is formed but in decreasing yield; larger complexes are not formed. The rate of complex formation decreases with increase of EYPC in the mixture. Reduction of pH in the reassembly mixture from 8.0 to 5.4 results in a marked reduction in complex formation indicating that ionized oleate facilitates lipidation. Removal of oleate by interaction of the complex with fatty acid-free human serum albumin does not degrade the complex. Incorporation of increasing amounts of unesterified cholesterol into the EYPC-sonicate progressively inhibits oleate-facilitated complex formation. This study shows that oleate, a physiologically relevant lipolysis-derived product, facilitates reassembly of apo A-I with EYPC and promotes formation of a small lipid-poor particle similar to that observed in nascent HDL and during in vivo or in vitro lipolysis of triacylglycerol-rich lipoproteins in the presence of HDL.

Structural and functional properties of human and mouse apolipoprotein A-I.

Mouse and human plasma apolipoprotein A-I (apo A-Im and apo A-Ih, respectively) were investigated to compare their molecular properties in solution, their incorporation into palmitoyloleoylphosphatidylcholine-apo A-I (POPC-apo A-I) discoidal complexes; their structural stability in discoidal complexes and high-density lipoproteins (HDL), and their effect on structural rearrangement of discoidal complexes upon interaction with low-density lipoproteins (LDL). Unlike apo A-Ih, only minimal concentration-dependent self-association was observed for apo A-Im. While both apo A-Im and apo A-Ih formed discoidal complexes of distinct composition and size that reflected reassembly molar ratios of POPC/apo A-I, apo A-Im demonstrated specific deficiencies in formation of larger-sized complexes. Denaturation of both apo A-Im- or apo A-Ih-containing complexes and HDL with guanidine hydrochloride (GuHCl) indicated significantly reduced stabilization of apo A-Im by lipid in these particles. Interaction of apo A-Im- or apo A-Ih-containing discoidal complexes with human plasma LDL revealed a more extensive conversion of apo A-Im-complexes to smaller species. Mean hydrophobicities and mean hydrophobic moments of amphipathic helical segments in apo A-Im and apo A-Ih were compared; differences potentially contributing to differential lipid-binding properties between apo A-Im and apo A-Ih were identified. Our results demonstrate differences between apo A-Im and apo A-Ih that may contribute to the major changes in plasma HDL distribution and function observed in apo A-Ih transgenic mice.

Apolipoprotein-lipid association in oxidatively modified HDL and LDL.

We investigated the effect of Cu2+ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation of HDL with Cu2+ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (lambda max). LDL incubated with Cu2+ also showed a rapid decrease in Trp fluorescence intensity with time, with no associated increase in lambda max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in lambda max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in lambda max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in lambda max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.

Protein composition determines the anti-atherogenic properties of HDL in transgenic mice.

High-density lipoprotein (HDL) contains two major proteins, apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II), comprising about 70% and 20% of the total HDL protein mass, respectively. HDL exists in human plasma in two main forms, one containing apoA-I with apoA-II (AI/AII-HDL) and another containing apoA-I without apoA-II (AI-HDL). A strong inverse relationship exists between total plasma HDL concentration and atherosclerosis, but the results of studies examining the relationship between AI-HDL and AI/AII-HDL and atherosclerosis have been conflicting. To determine whether these two HDL populations have different effects on atherogenesis, human apoA-I (AI) and human apoA-I and apoA-II (AI/AII) transgenic mice were produced in an atherosclerosis-susceptible strain. Following an atherogenic diet, despite similar total cholesterol and HDL cholesterol concentrations, the area of atherogenic lesions in the AI/AII mice was 15-fold greater than in the AI animals. These studies show that the protein composition of HDL significantly affects its role in atherogenesis and that AI-HDL is more antiatherogenic than AI/AII-HDL.

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.

Expression of human apolipoprotein A-II and its effect on high density lipoproteins in transgenic mice.

Apolipoproteins A-I and A-II comprise approximately 70 and 20%, respectively, of the total protein content of HDL. Evidence suggests that apoA-I plays a central role in determining the structure and plasma concentration of HDL, while the role of apoA-II is uncertain. To help define the function of apoA-II and determine what effect increasing its plasma concentration has on HDL, transgenic mice expressing human apoA-II and both human apoA-I and human apoA-II were produced. Human apoA-II mRNA is expressed exclusively in the livers of transgenic animals, and the protein exists as a dimer as it does in humans. High level expression of human apoA-II did not increase HDL concentrations or decrease plasma concentrations of murine apoA-I and apoA-II in contrast to what was observed in mice overexpressing human apoA-I. The primary effect of overexpressing human apoA-II was the appearance of small HDL particles composed exclusively of human apoA-II. HDL from mice transgenic for both human apoA-I and human apoA-II displayed a unique size distribution when compared with either apoA-I or apoA-II transgenic mice and contain particles with both these human apolipoproteins. These results in mice, indicating that human apoA-II participates in determining HDL size, parallel results from human studies.

Apolipoprotein AI expression and high density lipoprotein distribution in transgenic mice during development.

The developmental changes in apoAI expression in human apolipoprotein AI (apoAI) transgenic and nontransgenic control mice and the effect of these changes on high density lipoprotein (HDL) sizes were investigated. Results demonstrated that both human and mouse apoAI mRNA levels sharply increased following delivery and then decreased prior to weaning. Unlike the changes in apoAI mRNA, plasma apoAI concentration and HDL mass increased following delivery and remained elevated as the animals matured. In these animals, parallel increases in plasma apoAI and HDL mass with development were accompanied by increases in HDL particle sizes. Control mice demonstrated a monodisperse population of particles that increased in particle size from 7.5 nm (at 2 days prior to birth) to 9.8 nm (at 13 days after birth). The latter is similar to the adult HDL size and distribution. Transgenic mice, unlike controls, exhibited several distinct HDL populations, which also increased in size as animals matured. These results suggest that post-transcriptional factors are important in regulating apoAI plasma levels and that developmental changes in plasma apoAI concentrations are associated with changes in HDL size distribution.

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.

Evaluation of a method for study of kinetics of autologous apolipoprotein A-I.

Apolipoprotein A-I (apoA-I) participates in transport of plasma cholesterol. Concentrations of apoA-I depend on the balance between production and fractional clearance. To elucidate factors influencing apoA-I levels, accurate estimates of apoA-I turnover rates may be valuable. We describe a method for isolation of autologous apoA-I and its use in turnover studies. Free apoA-I was isolated from high density lipoproteins (HDL) by treatment with guanidine hydrochloride. This free apoA-I was radioiodinated with 131I and injected into eleven subjects simultaneously with HDL labeled with 125I. Plasma die-away curves of free apoA-I (131I) and HDL apoA-I (125I) were compared; fractional clearance rates averaged 0.256 +/- 0.019 (SEM) and 0.254 +/- 0.017 pools/day, respectively. Although slight differences between the two die-away curves were noted for some of the patients, the differences were relatively small; for the group as a whole, average fractional catabolic rates were not significantly different. Thus, by isolation of autologous apoA-I under the conditions described, free apoA-I seemingly provides a valid method for estimating apoA-I turnover.

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.

Apolipoprotein AIMilano. Disulfide-linked dimers increase high density lipoprotein stability and hinder particle interconversion in carrier plasma.

The in vitro metabolism of high density lipoproteins (HDL) in carriers of the apolipoprotein AIMilano (apoAIM) mutant was investigated during incubation of whole plasma and isolated lipoprotein fractions. A reduced cholesterol esterification (16.5 versus 25.0% for controls) and a decreased exchange of lipids between HDL and lower density lipoproteins was observed during incubation (6 h at 37 degrees C) of AIM plasma. Control HDL3 were converted to larger, faster-floating HDL particles, whereas only a fraction of AIM HDL3 followed the same pathway. Incubations were also carried out by mixing HDL3 from controls and AIM carriers with a lipoprotein-depleted plasma fraction in the presence of triglyceride-rich particles isolated from Intralipid. AIM HDL3 again showed a reduced capacity for lipid exchange; some HDL3 particles followed a "normal" conversion to faster-floating, larger HDL, whereas the small AIM HDL3 were not modified, indicating that AIM HDL3 are a mixture of metabolically functional and nonfunctional particles. Following transformation of the apoAIM homo- and heterodimers into their normal counterparts, i.e. monomeric apoAI and -AII, by reduction and carboxamidomethylation of AIM HDL3, the modified HDL3 behave like control HDL3 during incubation with lipoprotein-depleted plasma and triglyceride-rich particles. The presence of AIM dimers is most likely responsible for the increased HDL3 stability in the AIM carriers, indicating that apolipoprotein composition plays a major role in HDL particle interconversion.

Identifying the predominant peak diameter of high-density and low-density lipoproteins by electrophoresis.

Particle size distributions of high-density (HDL) and low-density (LDL) lipoproteins, obtained by polyacrylamide gradient gel electrophoresis, exhibit apparent predominant and minor peaks within characteristic subpopulation migration intervals. In the present report, we show that identification of such peaks as predominant or minor depends on whether the particle size distribution is analyzed according to migration distance or particle size. The predominant HDL peak on the migration distance scale is frequently not the predominant HDL peak when the distribution is transformed to the particle size scale. The potential physiologic importance of correct identification of the predominant HDL peak within a gradient gel electrophoresis profile is suggested by our cross-sectional study of 97 men, in which diameters associated with the predominant peak, determined using migration distance and particle size scales, were correlated with plasma lipoprotein and lipid parameters. Plasma concentrations of HDL-cholesterol, triglycerides, and apolipoproteins A-I and B correlated more strongly with the predominant peak obtained using the particle size scale than the migration distance scale. The mathematical transformation from migration distance to particle diameter scale had less effect on the LDL distribution. The additional computational effort required to transform the HDL-distribution into the particle size scale appears warranted given the substantial changes it produces in the gradient gel electrophoresis profile and the strengthening of correlations with parameters relevant to lipoprotein metabolism.

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.

Measurement of normative HDL subfraction cholesterol levels by Gaussian summation analysis of gradient gels.

This report describes development of a computerized method for analyzing polyacrylamide gradient gels of high density lipoproteins (HDL) by Gaussian summation, a simple technique to obtain standardized measurements of size and amount of HDL subfractions. Conditions for reproducibility and ranges of linearity were established. By Gaussian summation analysis, five or six HDL subfractions could be found in the plasma of most normolipidemic people. The relationship of staining intensity to cholesterol level was determined for Coomassie Blue R-250, permitting determination of the cholesterol levels in the individual subfractions, with standard errors of repeated measurements of 2% or less of the total HDL area, and accuracy, limited by the standard error of the chromogenicity, of 1-2 mg/dl for the least abundant fractions and 3-4 mg/dl for the most abundant subfractions. Levels of HDL2b measured by this method were statistically the same as levels of HDL2 measured by dextran sulfate-Mg2+ precipitation. Gaussian summation analysis of gradient gels was used to measure HDL subfraction cholesterol levels in subjects from the Baltimore Longitudinal Study on Aging to obtain normative levels for men and women for the major HDL subfractions. Comparisons of these levels with each other and with triglyceride and cholesterol levels showed that triglyceride levels were inversely correlated with levels of HDL2a and HDL2b, cholesterol levels were directly correlated with levels of HDL3b and HDL3a, and that HDL3b levels were inversely correlated with levels of both HDL2a and HDL2b.

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.