Biomedical subjects

K A Rye

Publications and source records attributed to K A Rye.

At least 37 records · Page 2Linked to original sources

PubMed1998

Factors influencing the ability of HDL to inhibit expression of vascular cell adhesion molecule-1 in endothelial cells.

We have previously reported that high density lipoproteins (HDLs) inhibit the cytokine-induced expression of adhesion molecules in endothelial cells. Here we investigate whether different preparations of HDLs vary in their ability to inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) activated by tumor necrosis factor-alpha (TNF-alpha). HDLs collected from a number of different human subjects all inhibited VCAM-1 expression in a concentration-dependent manner, although the extent of inhibition varied widely between subjects. The inhibitory activities of the HDL2 and HDL3 subfractions isolated from individual subjects also differed. Whether equated for concentrations of apolipoprotein (apo) A-I or cholesterol, the inhibitory activity of HDL3 was superior to that of HDL2. This difference remained apparent even when the HDL subfractions were present only during preincubations with the HUVECs and were removed before activation by TNF-alpha. To determine whether the inhibitory effect of HDL3 was influenced by apolipoprotein composition, preparations of HDL3 were modified by replacing all of their apo A-I with apo A-II. This change in apolipoprotein composition had no effect on the ability of the HDL3 to inhibit endothelial VCAM-1 expression. Thus, it has been shown that different preparations of HDLs differ markedly in their abilities to inhibit VCAM-1 expression in cytokine-activated HUVECs. The mechanism underlying the differences remains to be determined.

Apolipoprotein A-I↗

PubMed1998

Triglyceride-enrichment of high density lipoproteins enhances their remodelling by phospholipid transfer protein.

Phospholipid transfer protein (PLTP) converts high density lipoproteins (HDL) to larger and smaller particles during incubation in vitro. The present study shows that these conversions are enhanced in triglyceride (TG)-enriched HDL. When pig HDL were enriched with TG by incubation with cholesteryl ester transfer protein (CETP) and very low density lipoproteins, the TG/apoA-I molar ratio increased from 0.7/1 to 11.4/1 and the diameter increased from 9.0 to 9.5 nm. When unmodified pig HDL were incubated with PLTP, the diameter increased from 9.0 to 9.5 nm. Incubation of the TG-enriched pig HDL with PLTP generated larger (13.0 nm) and smaller (7.5 nm) particles. The composition of the large particles was comparable to the non-incubated, TG-enriched HDL. The small particles contained only phospholipids and apolipoprotein (apo) A-I. Studies were also carried out with spherical, reconstituted HDL (rHDL) containing only cholesteryl esters in their core. When the rHDL were enriched with TG by incubation with Intralipid and CETP, the diameter increased from 9.0 to 9.2 nm and the TG/apoA-I molar ratio became 5.7/1. When unmodified rHDL were incubated with PLTP, the diameter of 15% of the particles increased to 11.0 nm, 18% decreased to 7.6 nm, and 67% were unchanged. When the TG-enriched rHDL were incubated with PLTP, the diameter of 62% of the particles increased to 11.4 nm, 26% decreased to 7.6 nm, and 12% did not change. Preincubation with Intralipid alone or CETP alone did not enrich the rHDL with TG or enhance the size changes. These results show that the TG content of HDL has a major influence on PLTP-mediated size changes.

Animals↗

PubMed1997

Evidence that cholesteryl ester transfer protein-mediated reductions in reconstituted high density lipoprotein size involve particle fusion.

It is well established that cholesteryl ester transfer protein (CETP) changes the size of high density lipoproteins (HDL) during incubation in vitro. It has been suggested that HDL.CETP.HDL ternary complex formation is involved in these changes. The present results, which are consistent with CETP changing the size of spherical reconstituted HDL (rHDL) by a mechanism involving fusion, support the ternary complex hypothesis. When rHDL containing a core of cholesteryl esters and either three molecules of apolipoprotein (apo) A-I/particle, (A-I)rHDL, or six molecules of apoA-II/particle, (A-II)rHDL, were incubated individually with CETP, their respective diameters decreased from 9.4 to 7.8 nm and from 9.8 to 8.8 nm. The small (A-I)rHDL and (A-II)rHDL contained, respectively, two molecules of apoA-I/particle and four molecules of apoA-II/particle. As all of the rHDL lipids and apolipoproteins were quantitatively recovered at the end of the incubations, it was apparent that there was a 50% increase in the number of particles. This increase in the number of particles can be explained as follows: (i) sequential binding of two rHDL to CETP to generate a ternary complex, (ii) fusion of the rHDL in the ternary complex, and (iii) rearrangement of the fusion product into three small particles. Various spectroscopic techniques were used to show that the small rHDL were structurally distinct from the original rHDL. These results provide the first evidence that CETP mediates the fusion of spherical rHDL.

Carrier Proteins↗

PubMed1996

The influence of sphingomyelin on the structure and function of reconstituted high density lipoproteins.

The effect of sphingomyelin (SPM) on the structure and function of discoidal and spherical reconstituted high density lipoproteins (rHDL) has been studied. Three preparations of discoidal rHDL with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SPM/unesterified cholesterol (UC)/apolipoprotein (apo)A-I molar ratios of 99.6/0. 0/10.2/1.0, 86.0/13.6/10.8/1.0, and 72.5/26.3/11.4/1.0 were prepared by cholate dialysis. SPM did not affect discoidal rHDL size or surface charge. Esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT) was inhibited in the SPM-containing discoidal rHDL. When the discoidal rHDL of POPC/SPM/UC/apoA-I molar ratio 99.6/0.0/10.2/1.0 were incubated with low density lipoproteins (LDL) and LCAT, SPM transferred spontaneously from the LDL to the rHDL (t1/2 = 0.8 h) and spherical particles with a POPC/SPM/UC/CE/apoA-I molar ratio of 24.6/4.9/3. 6/24.9/1.0 were formed. Depleting the spherical rHDL of SPM head groups by incubation with sphingomyelinase increased the negative charge on the surface, but did not change their size. Cholesteryl ester transfer protein (CETP)-mediated transfers of cholesteryl esters and triglyceride between spherical rHDL and Intralipid were not affected by SPM head group depletion. The effect of SPM on rHDL structure was assessed spectroscopically. SPM increased POPC acyl chain and head group packing in the discoidal rHDL. When the spherical rHDL were depleted of SPM head groups, POPC acyl chain packing order decreased, but head group packing order was not affected. SPM inhibited the lipid-water interfacial hydration of discoidal rHDL. This parameter was not affected when the spherical rHDL were depleted of SPM head groups. The SPM molecule and the SPM head group, respectively, inhibited the unfolding of apoA-I in discoidal and spherical rHDL. It is concluded that (i) SPM influences the structure of discoidal and spherical rHDL, (ii) SPM inhibits the LCAT reaction in discoidal rHDL, and (iii) the SPM head group does not affect CETP-mediated lipid transfers into or out of spherical rHDL.

Carrier Proteins↗

PubMed1996

High density lipoproteins and coronary heart disease.

An inverse relationship between the concentration of high density lipoprotein (HDL) cholesterol and the development of coronary heart disease (CHD) is well established. It is unclear from the human studies whether this relationship reflects an ability of HDLs to protect against coronary disease or whether a low HDL in coronary patients is simply an epiphenomenon. Recent studies of transgenic mice, however, indicate that HDLs are directly antiatherogenic. The mechanism of the protection is unknown but may relate both to an involvement of HDLs in plasma cholesterol transport and to a range of non-lipid transport functions of HDLs. It is also unclear from human studies whether specific HDL subpopulations have differing abilities to protect against CHD, although such specificity is suggested from studies of transgenic mice. There is circumstantial evidence that elevating the concentration of HDL cholesterol in human subjects translates into a reduced coronary risk, although it should be stressed that there are still no reports of studies designed specifically to address this issue.

Animals↗

PubMed1996

Molecular mechanisms of reverse cholesterol transport.

The initial step of reverse cholesterol transport, the efflux of cell cholesterol into the extracellular fluid, has received much attention over the past year. Some studies have provided insights into the mechanism of cholesterol efflux and others have focused on the identity of the extracellular acceptors of cell cholesterol. Attention has also been directed towards the influence of HDL composition on the ability of these lipoproteins to accept cell cholesterol. There have been several studies addressing the importance of pre-beta-migrating HDL and of lipid-free apolipoproteins as acceptors of cell cholesterol. On the basis of these and earlier reports, a hypothetical cycle is proposed in which lipid-free apolipoprotein A-I dissociates from plasma HDL and transfers into the interstitial space where it acquires phospholipids and cholesterol from cells and becomes a discoidal HDL. Discoidal HDL transfer via lymphatics to the plasma where they are converted into mature HDL, thus completing the cycle.

Apolipoproteins↗

PubMed1996

Indirect sandwich enzyme-linked immunosorbent assay (ELISA) for plasma apolipoprotein E.

We have developed an indirect sandwich ELISA for measuring plasma apolipoprotein E (apo-E), using commercially available antibodies. A monoclonal anti-apo-E was used as the capture antibody and the captured apo-E detected with polyclonal anti-apo-E antiserum (goat). The detecting antibody was quantitated using horseradish peroxidase-conjugated rabbit immunoglobulin to goat immunoglobulins. There was no detectable cross-reactivity between the three antisera. Interference with the assay by apolipoprotein A-1, bilirubin and haemoglobin was not significant up to 1.7 g/L, 1250 mumol/L and 13.0 g/dL, respectively. The ELISA method showed high correlation with an established immunonephelometric method (ELISA apo.E = 1.04 Immunonephelometric apo-E + 16; r2 = 0.954, P < 0.0001, n = 39). The assay has a measuring range between 5 and 560 mg/L. The coefficient of duplicates was 2.0%, within-run coefficients of variation (CV) ranged from 3.7 to 6.0% and between-run CV from 6.1 to 15.1%. The reference range determined for 168 normotriglyceridaemic subjects was 20 to 130 mg/L. In an analysis of the lipoprotein subfractions isolated by ultracentrifugation as the fraction of density less than 1.25 g/mL and separated by gel permeation chromatography, apo-E was found to be associated with very low-density lipoprotein and large high-density lipoprotein.

Antibodies, Monoclonal↗

PubMed1996

A new molecular defect in the lecithin: cholesterol acyltransferase (LCAT) gene associated with fish eye disease.

We report a new genetic defect in the lecithin:cholesterol acyltransferase (LCAT) gene associated with classical clinical and biochemical features of fish eye disease. The 63-year-old Australian female proband also suffers from non-insulin-dependent (type II) diabetes mellitus. She presented with corneal opacities, markedly reduced HDL-cholesterol (0.1 mmol/L; < 10% of normal controls), and elevated plasma triglycerides. The presence of diabetes did not explain the lipoprotein profile, which differed markedly in comparison to two female hypertriglyceridemic diabetic subjects. Cholesterol esterification in HDL-like particles was minimal but plasma cholesterol esterification was maintained due to LCAT activity in non-HDL-containing lipoprotein fractions. DNA sequence analysis of the proband's LCAT gene showed two C to T transitions resulting in the substitution of Thr123 with Ile and Tyr144 with Cys. Allele-specific PCR amplification procedures were used to confirm the presence of the mutations in this proband and to screen for additional carriers in her family. Three first degree relatives (mother, brother, son) were heterozygous for the Thr123 --> Ile mutation and her daughter had the Tyr144 --> Cys mutation. Apart from a reduction in HDL-cholesterol levels to half the normal concentration and a 20% reduction in apoA-I levels, their plasma lipids were unremarkable. The proband's son and daughter were further investigated. Both had normal cholesterol esterification rates in plasma and VLDL/LDL-depleted plasma, but reduced LCAT activity (50% that of normal). Thus, the biochemical and phenotypic expression for fish eye disease in the heterozygote subjects was similar, irrespective of the underlying LCAT mutation.

Australia↗

PubMed1996

Remodelling of reconstituted high density lipoproteins by lecithin: cholesterol acyltransferase.

Discoidal reconstituted high density lipoproteins (rHDL) with a diameter of 7.9 nm, a molar ratio of egg phosphatidylcholine (PC): unesterified cholesterol (UC): cholesteryl esters (CE): apolipoprotein (apo) A-I of 33:7:0:1 and containing two molecules of apoA-1 per particle were incubated with lecithin:cholesterol acyltransferase (LCAT) in the presence of low density lipoproteins as a source of additional UC and PC for the LCAT reaction. After 24 h of incubation, the rHDL had a diameter of 8.8 nm, a molar ratio of PC:UC:CE:apoA-I of 16:3:23:1 and contained three rather than two molecules of apoA-I per particle. The fact that there was no change in the concentration of rHDL-associated apoA-I indicated that the increase from two to three molecules of apoA-I per particle was achieved at the expense of a one-third reduction in the number of rHDL particles in a process that must have involved particle fusion. When the incubations were repeated in the presence of exogenous, lipid-free apoA-I, the resulting rHDL were identical in size and composition to those generated in its absence. Under these conditions, however, the increase from two to three molecules of apoA-I per rHDL particle coincided with a 50% increase in the concentration of rHDL-associated apoA-I. Thus, when lipid-free apoA-I is available, the LCAT-mediated increase in number of apoA-I molecules per rHDL particle is achieved by a direct incorporation of lipid-free apolipoprotein without any need for particle fusion and therefore without a reduction in the number of rHDL particles.

Apolipoprotein A-I↗

PubMed1995

Cycling of apolipoprotein A-I between lipid-associated and lipid-free pools.

It has been shown previously that the reduction in particle size of HDL which follows incubation with the cholesteryl ester transfer protein (CETP) plus very-low-density lipoproteins (VLDL) or low-density lipoproteins (LDL) is accompanied by the dissociation of lipid-free apolipoprotein A-I (apo A-I) from HDL. In the present study, we demonstrate that this dissociation of apo A-I is reversible in a process dependent on the activity of lecithin:cholesterol acyltransferase (LCAT). The lipoprotein fraction (d < 1.21 g/ml) of human plasma was mixed with CETP and incubated under conditions such that the HDL decreased in size and there was a dissociation of about 30% of the apo A-I. Following this incubation, the d < 1.21 g/ml fraction was reisolated, supplemented with lipid-free apo A-I and reincubated in the presence and absence of LCAT, either as a component of lipoprotein-deficient plasma or as purified enzyme. In the absence of LCAT, HDL size did not increase and there was no incorporation of lipid-free apo A-I into the HDL density range. In contrast, when LCAT was present, the particle size of HDL increased and lipid-free apo A-I was incorporated into the HDL such that the HDL apo A-I content was comparable to that of the original, unmodified particles. The incorporation of lipid-free apo A-I into the HDL density range was dependent on both the presence of pre-existing HDL and an increase in their size. Thus, just as a reduction in HDL size is accompanied by the dissociation of lipid-free apo A-I, we have now shown that a subsequent increase in HDL size is accompanied by the reincorporation of lipid-free apo A-I into the particle.

Apolipoprotein A-I↗

PubMed1995

The influence of cholesteryl ester transfer protein on the composition, size, and structure of spherical, reconstituted high density lipoproteins.

The effect of cholesteryl ester transfer protein (CETP) on the size, composition, and structure of spherical, reconstituted HDL (rHDL) which contain apolipoprotein (apo) A-I as their sole apolipoprotein has been studied. Spherical rHDL were incubated with CETP and Intralipid for up to 24 h. During this time CETP promoted transfers of cholesteryl esters (CE) and triglyceride (TG) between rHDL and Intralipid. As a result, the rHDL became depleted of CE and enriched in TG. However, as the loss of CE from the rHDL was greater than the gain of TG, the concentration of core lipids in the rHDL decreased. The decrease in the concentration of rHDL core lipids, which was evident throughout the incubation, was accompanied by a reduction in rHDL diameter from 9.2 to 8.0 nm, the dissociation of apoA-I from rHDL and a decrease in the number of apoA-I molecules, from three/particle in the 9.2-nm rHDL, to two/particle in the 8.0-nm rHDL. Spectroscopic studies showed that the lipid-water interface and phospholipid packing of the 8.0-nm rHDL were, respectively, more polar and less ordered than those of the 9.2-nm rHDL. Quenching studies with KI revealed that the number of exposed apoA-I Trp residues in the 9.2- and 8.0-nm rHDL was two and three, respectively. Circular dichroism established that the 9.2- and 8.0-nm rHDL had identical apoA-I alpha-helical contents. The 9.2- and 8.0-nm rHDL also had identical surface charges as determined by agarose gel electrophoresis. Denaturation studies with guanidine hydrochloride demonstrated that apoA-I is more stable in 8.0-nm rHDL than in 9.2-nm rHDL. It is concluded that CETP converts rHDL to small, TG-enriched, apoA-I-depleted particles with increased lipid-water interfacial hydration and less ordered phospholipid packing. These changes are associated with enhanced stability and minor changes to the conformation of the apoA-I which remains associated with the rHDL.

Apolipoprotein A-I↗

PubMed1995

High-density lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules.

While an elevated plasma concentration of HDLs is protective against the development of atherosclerosis and ensuing coronary heart disease (CHD), the mechanism of this protection is unknown. One early cellular event in atherogenesis is the adhesion of mononuclear leukocytes to the endothelium. This event is mediated principally by vascular cell adhesion molecule-1 (VCAM-1) but also involves other molecules, such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin. We have investigated the effect of isolated plasma HDLs and reconstituted HDLs on the expression of these molecules by endothelial cells. We show that physiological concentrations of HDLs inhibit tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 (IL-1) induction of these leukocyte adhesion molecules in a concentration-dependent manner. Steady state mRNA levels of TNF-alpha-induced VCAM-1 and E-selectin are significantly reduced by physiological concentrations of HDLs. An an HDL concentration of 1 mg/mL apolipoprotein A-I, the protein expressions of VCAM-1, ICAM-1, and E-selectin were inhibited by 89.6 +/- 0.4% (mean +/-SD, n=4), 64.8 +/- 1.0%, and 79.2 +/- 0.4%, respectively. In contrast, HDLs have no effect on the expression of platelet endothelial cell adhesion molecule (PECAM) or on the expression of the p55 and p75 subunits of the TNF-alpha receptor. HDLs were effective when added from 16 hours before to 5 minutes after cytokine stimulation. HDLs had no effect on TNF-alpha-induced expression of ICAM-1 by human foreskin fibroblasts, suggesting that the effect is cell-type restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

PubMed1995

Exchange of oxidized cholesteryl linoleate between LDL and HDL mediated by cholesteryl ester transfer protein.

This study examines the cholesteryl ester transfer protein (CETP)-mediated exchange of cholesteryl linoleate hydroperoxide (Ch18:2-OOH) and cholesteryl linoleate hydroxide (Ch18:2-OH) between low density lipoprotein (LDL) and high density lipoprotein (HDL). When [3H]Ch18:2-OOH- and [3H]18:2-OH-labeled LDL were incubated at 37 degrees C for 0-24 h with unoxidized HDL and purified CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the HDL. Similarly, when incubations were carried out with [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled HDL, unoxidized LDL, and CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the LDL. Comparable results were obtained for the CETP-mediated transfer of [3H]Ch18:2-OH alone from LDL to HDL. Transfer to HDL of oxidized cholesteryl linoleate from [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled LDL was comparable to that of unoxidized cholesteryl linoleate (Ch18:2). However, the rate of transfer of [3H]Ch18:2-OOH and [3H]Ch18:2-OH from LDL to HDL increased linearly as the molar ratio of acceptor (HDL) to donor (oxidized LDL) particles in the incubation increased from 0.5:1 to 10:1. This increased rate of exchange was accompanied by an increased proportion of the oxidized Ch18:2 being present as the hydroxide rather than hydroperoxide. Further increases in the molar ratio of HDL to oxidized LDL particles neither affected the transfer rate nor the extent of reduction of Ch18:2-OOH to Ch18:2-OH. We therefore conclude that i) CETP mediates bidirectional transfers of Ch18:2-OOH and Ch18:2-OH between HDL and LDL; ii) CETP does not distinguish between Ch18:2-OOH, Ch18:2-OH, and Ch18:2 as it mediates their exchange between HDL and LDL; and iii) association with HDL hastens the reduction of Ch18:2-OOH to Ch18:2-OH.

Adult↗

PubMed1994

The influence of apolipoproteins on the structure and function of spheroidal, reconstituted high density lipoproteins.

This study investigates the influence of apolipoproteins on the structure and function of spheroidal, reconstituted high density lipoproteins (rHDL). Spheroidal rHDL containing apolipoprotein (apo) A-I, (A-I)rHDL, were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. Spheroidal rHDL containing apoA-II, (A-II)rHDL, were prepared by displacing apoA-I from (A-I)rHDL with apoA-II. When the (A-I)rHDL were subjected to agarose gel electrophoresis, their migration was retarded relative to native HDL. The (A-II)rHDL migrated slower than either the (A-I)rHDL or native HDL. Spectroscopic studies showed that the packing order of the rHDL phospholipids was independent of apolipoprotein composition and that the polarity, or hydration, of the lipid-water interface of the (A-I)rHDL was greater than that of the (A-II)rHDL. When the rHDL were incubated with very low density lipoproteins (VLDL) and cholesteryl ester transfer protein, comparable amounts of cholesteryl ester (CE) were transferred from (A-I)rHDL and (A-II)rHDL to VLDL. The transfer of triglyceride (TG) from VLDL to (A-I)rHDL and (A-II)rHDL was also comparable. Similar results were obtained when Intralipid was substituted for VLDL. It is concluded that (i) apolipoproteins influence the surface charge of spheroidal rHDL, (ii) the hydration of the lipid-water interface of (A-I)rHDL is greater than that of (A-II)rHDL and (ii) cholesteryl ester transfer protein-mediated transfers of CE and TG between spheroidal rHDL and TG-rich particles are independent of rHDL apolipoprotein composition.

Apolipoproteins↗

PubMed1994

Cholesteryl ester transfer protein: its role in plasma lipid transport.

1. The cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein which acts in plasma to redistribute cholesteryl esters and triglyceride between plasma lipoproteins. 2. CETP also plays an important role in determining the composition and particle size distribution of high density lipoproteins (HDL). 3. Activity of CETP may be regulated in four ways: By factors which influence the concentration of CETP in plasma; by the activity of CETP inhibitor proteins; by variations in the concentrations and compositions of donor and acceptor lipoproteins and by factors which influence the interaction of CETP with plasma lipoproteins. 4. The mechanism of action of CETP is uncertain. Two models have been proposed: (i) a shuttle model in which CETP physically transports lipids between lipoprotein particles and (ii) a ternary complex model in which CETP forms a bridge between two lipoprotein particles, enabling them to exchange lipids. 5. Evidence is accumulating that CETP may be a pro-atherogenic factor.

Animals↗

PubMed1994

High-density lipoproteins and coronary heart disease.

High-density lipoproteins (HDLs) play an important role in the process of reverse cholesterol transport, the pathway by which the cholesterol in extrahepatic tissues is transported through plasma to the liver for recycling or for excretion from the body in bile. The concentration of HDL cholesterol is a powerful inverse predictor of the development of coronary heart disease, leading to a widely held view that HDL protects against the development of atherosclerosis. The mechanism by which HDLs protect is unknown. To date, no studies have been designed specifically to test the proposition that increasing the concentration of HDL cholesterol translates into a reduction in coronary risk. Nevertheless, in a subgroup of the Helsinki Heart Study, it was found that a substantial proportion of the beneficial effect of gemfibrozil was explicable in terms of an increase in the concentration of HDL cholesterol.

Coronary Disease↗

PubMed1994

Dissociation of lipid-free apolipoprotein A-I from high density lipoproteins.

Conditions under which apolipoprotein (apo) A-I dissociates from human high density lipoproteins (HDL) during incubation in vitro have been investigated. Dissociation of apoA-I was demonstrated by non-denaturing gradient gel electrophoresis followed by immunoblotting for apoA-I and by size-exclusion chromatography. It was quantitated after ultracentrifugation as the loss of apoA-I from the fraction of d < 1.25 g/ml. ApoA-I did not dissociate from HDL when they were incubated alone at 37 degrees C for up to 24 h. Nor was there dissociation of apoA-I when the HDL were incubated either with the cholesteryl ester transfer protein (CETP) in the absence of other lipoprotein fractions or with other lipoproteins in the absence of CETP. However, when mixtures of HDL and CETP were incubated for 24 h in the presence of physiological concentrations of either very low density lipoproteins (VLDL) or low density lipoproteins (LDL), there was a dissociation of up to 36% of the apoA-I from the HDL fraction that was linear with time. The dissociation of apoA-I coincided with a time-dependent reduction in HDL particle size. The percentage of apoA-I that dissociated from HDL correlated positively with the concentrations of VLDL, LDL, and CETP in the incubation but negatively with the concentration of HDL. When lecithin:cholesterol acyltransferase was added to mixtures at the completion of 24 h of incubation with CETP, the size of the HDL increased and the dissociated apoA-I returned to the fraction of d < 1.25 g/ml. analysis of the lipoprotein-deficient fraction of d > 1.25 g/ml isolated by ultracentrifugation and of the lower molecular weight fractions recovered after size-exclusion chromatography revealed that the dissociated apoA-I was not associated with significant quantities of either cholesterol, phospholipids, or other apolipoproteins. When the dissociated apoA-I was subjected to agarose gel electrophoresis, it migrated to a prebeta position comparable to that of purified, lipid-free apoA-I. This contrasted with the original HDL that exhibited alpha migration. Thus, CETP-mediated transfers of cholesteryl esters from HDL to VLDL and LDL are accompanied not only by a reduction in HDL size but also by the progressive dissociation from HDL of a pool of prebeta-migrating, essentially lipid-free apoA-I.

Apolipoprotein A-I↗

PubMed1993

Preparation and characterization of spheroidal, reconstituted high-density lipoproteins with apolipoprotein A-I only or with apolipoprotein A-I and A-II.

This study describes the preparation of spheroidal reconstituted HDL which contain apolipoprotein (apo) A-I only, (A-I w/o A-II) r-HDL, or apo A-I and apo A-II, (A-I w A-II) r-HDL. Spheroidal (A-I w/o A-II) r-HDL with diameters of 8.0, 9.2 and 11.2 nm were prepared by incubating discoidal (A-I w/o A-II) r-HDL with lecithin-cholesterol acyltransferase and low-density lipoproteins. Spheroidal (A-I w A-II) r-HDL were prepared by displacing apo A-I from spheroidal (A-I w/o A-II) r-HDL with apo A-II. Modification with apo A-II did not significantly affect the diameters of the 8.0 and 9.2 nm (A-I w/o A-II) r-HDL. When, however, apo A-II was added to the (A-I w/o A-II) r-HDL of diameter 11.2 nm, the size of the particles decreased to 9.4 nm. To determine whether modification of (A-I w/o A-II) r-HDL with apo A-II altered the structure of the r-HDL, the packing of phospholipids in the modified and unmodified particles was compared by steady state fluorescence polarization and the environments of the apo A-I tryptophan residues in (A-I w/o A-II) and (A-I w A-II) r-HDL were compared by fluorescence spectroscopy. The results of these studies suggested that modification of spheroidal (A-I w/o A-II) r-HDL with apo A-II alters the environment of apo A-I tryptophan residues in small, but not large, r-HDL and does not affect the packing of phospholipids.

Apolipoprotein A-I↗