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Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.

Syndromes associated with LCAT deficiency, a rare autosomal recessive condition, include fish-eye disease (FED) and familial LCAT deficiency (FLD). FLD is more severe and characterized by early and progressive chronic kidney disease (CKD). No treatment is currently available for FLD, but novel therapeutics are under development. Furthermore, although biomarkers of LCAT deficiency have been identified, their suitability to monitor disease progression and therapeutic efficacy is unclear, as little data exist on the rate of progression of renal disease. Here, we systematically review observational studies of FLD, FED, and heterozygous subjects, which summarize available evidence on the natural history and biomarkers of LCAT deficiency, in order to guide the development of novel therapeutics. We identified 146 FLD and 53 FED patients from 219 publications, showing that both syndromes are characterized by early corneal opacity and markedly reduced HDL-C levels. Proteinuria/hematuria were the first signs of renal impairment in FLD, followed by rapid decline of renal function. Furthermore, LCAT activity toward endogenous substrates and the percentage of circulating esterified cholesterol (EC%) were the best discriminators between these two syndromes. In FLD, higher levels of total, non-HDL, and unesterified cholesterol were associated with severe CKD. We reveal a nonlinear association between LCAT activity and EC% levels, in which subnormal levels of LCAT activity were associated with normal EC%. This review provides the first step toward the identification of disease biomarkers to be used in clinical trials and suggests that restoring LCAT activity to subnormal levels may be sufficient to prevent renal disease progression.

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20–200 mg/dl FC; coefficient of variation

We assessed secondary and genetic causes of severe HDL deficiency in 258,252 subjects, of whom 370 men (0.33%) and 144 women (0.099%) had HDL cholesterol levels G; 2) LCAT (12.4%): 1 homozygote, 3 compound heterozygotes, 13 heterozygotes, and 8 heterozygotes with variant rs4986970/p.S232T; 3) APOA1 (5.0%): 1 homozygote and 9 heterozygotes; and 4) LPL (4.5%): 1 heterozygote and 8 heterozygotes with variant rs268/p.N318S. In addition, 4.5% had other mutations, and 46.8% had no mutations. Atherosclerotic cardiovascular disease (ASCVD) prevalence rates in the ABCA1,LCAT,APOA1, LPL, and mutation-negative groups were 37.0%, 4.0%, 40.0%, 11.1%, and 6.4%, respectively. Severe HDL deficiency is uncommon, with 40.1% having secondary causes and 48.8% of the subjects sequenced having ABCA1,LCAT,APOA1, or LPL mutations or variants, with the highest ASCVD prevalence rates being observed in the ABCA1 and APOA1 groups.

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL';s cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1';s ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P< 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5–7 on one APOA1 molecule and helices 3–4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

The aim of this study was to evaluate the vasoprotective effects of HDL isolated from carriers of LCAT deficiency, which are characterized by a selective depletion of LpA-I:A-II particles and predominance of preβ migrating HDL. HDLs were isolated from LCAT-deficient carriers and tested in vitro for their capacity to promote NO production and to inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in cultured endothelial cells. HDLs from carriers were more effective than control HDLs in promoting eNOS activation with a gene-dose-dependent effect (PTrend = 0.048). As a consequence, NO production induced by HDL from carriers was significantly higher than that promoted by control HDL (1.63 ± 0.24-fold vs. 1.34 ± 0.07-fold, P= 0.031). HDLs from carriers were also more effective than control HDLs in inhibiting the expression of VCAM-1 (homozygotes, 65.0 ± 8.6%; heterozygotes, 53.1 ± 7.2%; controls, 44.4 ± 4.1%; PTrend = 0.0003). The increased efficiency of carrier HDL was likely due to the depletion in LpA-I:A-II particles. The in vitro findings might explain why carriers of LCAT deficiency showed flow-mediated vasodilation and plasma-soluble cell adhesion molecule concentrations comparable to controls, despite low HDL-cholesterol levels. These results indicate that selective depletion of apoA-II-containing HDL, as observed in carriers of LCAT deficiency, leads to an increased capacity of HDL to stimulate endothelial NO production, suggesting that changes in HDL apolipoprotein composition may be the target of therapeutic interventions designed to improve HDL functionality.

Lipoproteins play a key role in the development of CVD, but the dynamics of lipoprotein metabolism are difficult to address experimentally. This article describes a novel two-step combined in vitro and in silico approach that enables the estimation of key reactions in lipoprotein metabolism using just one blood sample. Lipoproteins were isolated by ultracentrifugation from fasting plasma stored at 4°C. Plasma incubated at 37°C is no longer in a steady state, and changes in composition may be determined. From these changes, we estimated rates for reactions like LCAT (56.3 µM/h), β-LCAT (15.62 µM/h), and cholesteryl ester (CE) transfer protein-mediated flux of CE from HDL to IDL/VLDL (21.5 µM/h) based on data from 15 healthy individuals. In a second step, we estimated LDL's HL activity (3.19 pools/day) and, for the very first time, selective CE efflux from LDL (8.39 µM/h) by relying on the previously derived reaction rates. The estimated metabolic rates were then confirmed in an independent group (n = 10). Although measurement uncertainties do not permit us to estimate parameters in individuals, the novel approach we describe here offers the unique possibility to investigate lipoprotein dynamics in various diseases like atherosclerosis or diabetes.

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A2 (PLA2) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA2. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer's disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT−/− mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer's-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.

In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P < 0.001) and complete HDL-cholesteryl ester deficiency. ApoB-containing lipoprotein and associated triglyceride levels are increased in LCAT KO mice as compared with C57BL/6 control mice (44%; P < 0.05). Glucocorticoid-producing adrenocortical cells within the zona fasciculata in LCAT KO mice are devoid of neutral lipids. However, adrenal weights and basal corticosterone levels are not significantly changed in LCAT KO mice. In contrast, adrenals of LCAT KO mice show compensatory up-regulation of genes involved in cholesterol synthesis (HMG-CoA reductase; 516%; P < 0.001) and acquisition (LDL receptor; 385%; P < 0.001) and a marked 40–50% lower glucocorticoid response to adrenocorticotropic hormone exposure, endotoxemia, or fasting (P < 0.001 for all). In conclusion, our studies show that HDL-cholesteryl ester deficiency in LCAT KO mice is associated with a 40–50% lower adrenal glucocorticoid output. These findings further highlight the important novel role for HDL as cholesterol donor for the synthesis of glucocorticoids by the adrenals.

The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I−/− mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-β-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I−/−× apoE−/− mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-β-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1−/− or LCAT−/− mice. Coexpression of apoA-IV and LCAT in apoA-I−/− mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.

Lipid transfer inhibitor protein (LTIP) exists in both active and inactive forms. Incubation (37°C) of plasma causes LTIP to transfer from a 470 kDa inactive complex to LDL where it is active. Here, we investigate the mechanisms underlying this movement. Inhibiting LCAT or cholesteryl ester transfer protein (CETP) reduced incubation-induced LTIP translocation by 40–50%. Blocking both LCAT and CETP completely prevented LTIP movement. Under appropriate conditions, either factor alone could drive maximum LTIP transfer to LDL. These data suggest that chemical modification of LDL, the 470 kDa complex, or both facilitate LTIP movement. To test this, LDL and the 470 kDa fraction were separately premodified by CETP and/or LCAT activity. Modification of the 470 kDa fraction had no effect on subsequent LTIP movement to native LDL. Premodification of LDL, however, induced spontaneous LTIP movement from the native 470 kDa particle to LDL. This transfer depended on the extent of LDL modification and correlated negatively with changes in the LDL phospholipid + cholesterol-to-cholesteryl ester + triglyceride ratio. We conclude that LTIP translocation is dependent on LDL lipid composition, not on its release from the inactive complex. Compositional changes that reduce the surface-to-core lipid ratio of LDL promote LTIP binding and activation.

We have identified a novel mutation in apoA-I (serine 36 to alanine; S36A) in a human subject with severe hypoalphalipoproteinemia. The mutation is located in the N-terminal region of the protein, which has been implicated in several functions, including lipid binding and lecithin:cholesterol acyltransferase (LCAT) activity. In the present study, the S36A protein was produced recombinantly and characterized both structurally and functionally. While the helical content of the mutant protein was lower compared with wild-type (WT) apoA-I, it retained its helical character. The protein stability, measured as the resistance to guanidine-induced denaturation, decreased significantly. Interestingly, native gel electrophoresis, cross-linking, and sedimentation equilibrium analysis showed that the S36A mutant was primarily present as a monomer, notably different from the WT protein, which showed considerable oligomeric forms. Although the ability of S36A apoA-I to solubilize phosphatidylcholine vesicles and bind to lipoprotein surfaces was not altered, a significantly impaired LCAT activation compared with the WT protein was observed. These results implicate a region around S36 in apoA-I self-association, independent of the intact C terminus. Furthermore, the region around S36 in the N-terminus of human apoA-I is necessary for LCAT activation.

The specifics of nascent HDL remodeling within the plasma compartment remain poorly understood. We developed an in vitro assay to monitor the lipid transfer between model nascent HDL (LpA-I) and plasma lipoproteins. Incubation of α-125I-LpA-I with plasma resulted in association of LpA-I with existing plasma HDL, whereas incubation with TD plasma or LDL resulted in conversion of α-125I-LpA-I to preβ-HDL. To further investigate the dynamics of lipid transfer, nascent LpA-I were labeled with cell-derived [3 H]cholesterol (UC) or [3H]phosphatidylcholine (PC) and incubated with plasma at 37°C. The majority of UC and PC were rapidly transferred to apolipoprotein B (apoB). Subsequently, UC was redistributed to HDL for esterification before being returned to apoB. The presence of a phospholipid transfer protein (PLTP) stimulator or purified PLTP promoted PC transfer to apoB. Conversely, PC transfer was abolished in plasma from PLTP−/− mice. Injection of 125I-LpA-I into rabbits resulted in a rapid size redistribution of 125I-LpA-I. The majority of [3H]UC from labeled r(HDL) was esterified in vivo within HDL, whereas a minority was found in LDL. These data suggest that apoB plays a major role in nascent HDL remodeling by accepting their lipids and donating UC to the LCAT reaction. The finding that nascent particles were depleted of their lipids and remodeled in the presence of plasma lipoproteins raises questions about their stability and subsequent interaction with LCAT.

We investigated the in vivo metabolic fate of pre-β HDL particles in human apolipoprotein A-I transgenic (hA-ITg) mice. Pre-β HDL tracers were assembled by incubation of [125I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-β HDLs of increasing size (pre-β1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-ITg-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-β2, -3, and -4 had similar plasma die-away rates, whereas pre-β1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-β HDL size increased. In plasma, pre-β1 and -2 were rapidly (

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI−/−) macrophages. Briefly cultured macrophages collected from SR-BI−/− mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI−/− mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI−/− bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-I Tg SR-BI−/−) mice. Compared with hA-I Tg mice, hA-I Tg SR-BI−/− mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13–18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-I Tg SR-BI−/− mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-I Tg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-I Tg SR-BI−/− mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

The plasma HDLs represent a major class of cholesterol-transporting lipoprotein that can be divided into two distinct subfractions, HDL2 and HDL3, by ultracentrifugation. Existing methods for the subfractionation of HDL requires lengthy ultracentrifugations, making them unappealing for large-scale studies. We describe a method that subfractionates HDL from plasma in only 6 h, representing a substantial decrease in total isolation time. The subfractions so isolated were assessed for a variety of lipid and protein components, in addition to their susceptibility to oxidation, both alone and in combination with VLDL and LDL. We report for the first time a prooxidant role for HDL during VLDL oxidation, in which HDL donates preformed hydroperoxides to VLDL in a cholesteryl ester transfer protein (CETP)-dependent process. Examination of the participation of HDL in LDL oxidation has reinforced its classic role as a potent antioxidant. Furthermore, we have also implicated the second major HDL-associated enzyme, LCAT, in these processes, whereby it acts as a potent prooxidant during VLDL oxidation but as an antioxidant during LDL oxidation. Thus, we have identified a potentially duplicitous role for HDL in the pathogenesis of atherosclerosis, attributable to both CETP and LCAT.

The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 ± 2.1% vs. 3.9 ± 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 ± 3.2% vs. 66.7 ± 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 ± 4.0% vs. 54.6 ± 1.9%) and significantly increased the phospholipid percentage (11.2 ± 0.9% vs. 19.3 ± 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 ± 0.8% in controls to 0 ± 0.7% in ACAT2-deficient mice.In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.

Val156 of apolipoprotein A-I (apoA-I) was found to be a key amino acid in the structure and function of high density lipoprotein (HDL) (J. Biol. Chem., 275: 26821–26827, 2000). To determine more precisely the functions of the individual amino acids proximal to Val156, serial point mutants of proapoA-I, including V156K, D157K, and A158E, were overexpressed and purified to at least 95% purity. In the lipid-free state, A158E exhibited the most profound self-associative patterns and the least pronounced dimyristoyl phosphatidylcholine (DMPC) clearance activities. In the lipid-bound state, A158E formed a larger reconstituted HDL (rHDL) with palmitoyloleoyl phosphatidylcholine (POPC), ∼120 Å, whereas other mutants and the wild type (WT) formed 97 Å of POPC-rHDL. Cross-linking analysis revealed that A158E-rHDL harbored at least four protein molecules in the particle, while other rHDL conformations contained only two protein molecules. All of the POPC-rHDL produced smaller HDL, around 78 Å, after 24 h of incubation in the presence of low density lipoprotein at 37°C. V156K and A158E exhibited decreased lecithin:cholesterol acyltransferase activation activity in the POPC-rHDL state, showing

The percentage of saturated cholesteryl esters (CEs) synthesized by human LCAT is several times higher than expected from the sn-2 acyl composition of plasma phosphatidylcholine (PC), whereas the synthesis of 20:4 CE and 22:6 CE is much lower than expected. To explain these discrepancies, we proposed that LCAT transfers some saturated fatty acids from the sn-1 position of PC species that contain 20:4 or 22:6 at sn-2. The present studies provide in vivo evidence for this hypothesis. We determined the composition and synthesis of CE species in plasma of volunteers before and after a 6 week dietary supplementation with docosahexaenoic acid (22:6; DHA). In addition to an increase in the DHA content of all plasma lipids, there was a significant (+12%; P < 0.005) increase of 16:0 CE, although there was no increase in 16:0 at sn-2 of PC. The increase of DHA in CE was much lower than its increase at sn-2 of PC. Ex vivo synthesis of CE species in plasma showed a significant (+24%; P < 0.005) increase in the synthesis of 16:0 CE after DHA supplementation, which correlated positively with the increase of 22:6, but not of 16:0, at sn-2 of PC.These results show that the positional specificity of human LCAT is altered when the concentration of 16:0-22:6 PC is increased by DHA supplementation.

We examined whether postprandial (PP) chylomicrons (CMs) can serve as vehicles for transporting cholesterol from endogenous cholesterol-rich lipoprotein (LDL+HDL) fractions and cell membranes to the liver via lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities. During incubation of fresh fasting and PP plasma containing [3H]cholesteryl ester (CE)-labeled LDL+HDL, both CMs and VLDL served as acceptors of [3H]CE or cholesterol from LDL+HDL. The presence of CMs in PP plasma suppressed the ability of VLDL to accept [3H]CE from LDL+HDL. In reconstituted plasma containing an equivalent amount of triglycerides from isolated VLDL or CMs, a CM particle was about 40 times more potent than a VLDL particle in accepting [3H]CE or cholesterol from LDL+HDLs. When incubated with red blood cells (RBCs) as a source for cell membrane cholesterol, the cholesterol content of CMs, VLDL, LDL, and HDL in PP plasma increased by 485%, 74%, 13%, and 30%, respectively, via LCAT and CETP activities. The presence of CMs in plasma suppressed the ability of endogenous lipoproteins to accept cholesterol from RBCs.Our data suggest that PP CMs may play an important role in promoting reverse cholesterol transport in vivo by serving as the preferred ultimate vehicle for transporting cholesterol released from cell membranes to the liver via LCAT and CETP.

The effect of apolipoprotein A-II (apoA-II) on the structure and stability of HDL has been investigated in reconstituted HDL particles. Purified human apoA-II was incorporated into sonicated, spherical LpA-I particles containing apoA-I, phospholipids, and various amounts of triacylglycerol (TG), diacylglycerol (DG), and/or free cholesterol. Although the addition of PC to apoA-I reduces the thermodynamic stability (free energy of denaturation) of its α-helices, PC has the opposite effect on apoA-II and significantly increases its helical stability. Similarly, substitution of apoA-I with various amounts of apoA-II significantly increases the thermodynamic stability of the particle α-helical structure. ApoA-II also increases the size and net negative charge of the lipoprotein particles. ApoA-II directly affects apoA-I conformation and increases the immunoreactivity of epitopes in the N and C termini of apoA-I but decreases the exposure of central domains in the molecule (residues 98–186). ApoA-II appears to increase HL association with HDL and inhibits lipid hydrolysis. ApoA-II mildly inhibits PC hydrolysis in TG-enriched particles but significantly inhibits DG hydrolysis in DG-rich LpA-I. In addition, apoA-II enhances the ability of reconstituted LpA-I particles to inhibit VLDL-TG hydrolysis by HL.Therefore, apoA-II affects both the structure and the dynamic behavior of HDL particles and selectively modifies lipid metabolism.

We have established an immunoassay for preβ1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because preβ1-HDL is unstable during storage, fresh plasma must be used for preβ1-HDL measurements. In this study, we describe a method of stabilizing preβ1-HDL, and evaluate the analytical performance of the immunoassay for preβ1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Preβ1-HDL concentration was measured by immunoassay. In nonpretreated samples, preβ1-HDL decreased significantly from the baseline after 6 h at room temperature. Although preβ1-HDL was more stable at 0°C than at room temperature, it increased from 30.2 ± 8.5 (SE) to 56.5 ± 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 ± 1.2 to 37.9 ± 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at −80°C, preβ1-HDL increased from 29.0 ± 4.0 to 38.0 ± 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, preβ1-HDL concentration did not change significantly under any of the above conditions. Moreover, preβ1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05).An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of preβ1-HDL.

Postprandial triglyceride-rich lipoproteins (TRL) exert proatherogenic effects at the arterial wall, including lipid deposition. Following consumption of a mixed meal (1,200 kcal), plasma-mediated cellular free cholesterol (FC) efflux, lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP) activities were determined in subjects (n = 12) displaying type IIB hyperlipidemia and compared with those in a normolipidemic control group (n = 14). The relative capacity of plasma to induce FC efflux from Fu5AH cells via the SR-BI receptor was significantly increased 4 h postprandially (+23%; P < 0.005) in the type IIB group, whereas it remained unchanged for postprandial plasma from normolipidemic subjects. LCAT activity was significantly elevated 2 h postprandially in both the IIB and control groups, (+46% and +36%, respectively; P < 0.005 vs. respective baseline value). In type IIB subjects, total cholesteryl ester (CE) mass transfer from HDL to total TRL [chylomicrons (CMs) + VLDL-1 + VLDL-2 + IDL] increased progressively from 15 ± 2 μg CE/h/ml at baseline to 28 ± 2 μg CE transferred/h/ml (+87%; P = 0.0004) at 4 h postprandially. CE transfer to CMs and VLDL-1 was preferentially stimulated (2.6-fold and 2.3-fold respectively) at 4 h in IIB subjects and occurred concomitantly with elevation in mass and particle number of both CMs (2.3-fold) and VLDL-1 (1.3-fold). Furthermore, in type IIB subjects, CETP-mediated total CE flux over the 8 h postprandial period from HDL to potentially atherogenic TRL was significantly enhanced, and notably to VLDL-1 (32-fold elevation; P < 0.005), relative to control subjects. Such CE transfer flux was reflected in a significant postprandial increase in CE-TG ratio in both CMs and VLDL-1 in type IIB plasmas.In conclusion, HDL-CE is preferentially targeted to VLDL-1 via the action of CETP during alimentary lipemia, thereby favoring formation and accumulation of atherogenic CE-rich remnant particles.

Hypertriglyceridemia induces multiple changes in lipoprotein composition. Here we investigate how one of these modifications, triglyceride (TG) enrichment, affects HDL and LDL function when this alteration occurs under conditions in which more polar components can naturally re-equilibrate. TG-enriched lipoproteins were produced by co-incubating VLDL, LDL, and HDL with cholesteryl ester (CE) transfer protein. The resulting 2.5-fold increase in TG/CE ratio did not measurably alter the apoprotein composition of LDL or HDL, or modify LDL size. HDL mean diameter increased slightly from 9.1 to 9.4 nm. Modified LDL was internalized by fibroblasts normally, but its protein was degraded much less efficiently. This likely reflects an aberrant apolipoprotein B (apoB) conformation, as suggested by its resistance to V8 protease digestion and altered LDL electrophoretic mobility. TG-enriched LDL ineffectively down-regulated cholesterol biosynthesis compared with control LDL at the same protein concentration, but was equivalent in sterol regulation when compared on a cholesterol basis. TG-enriched HDL promoted greater net cholesterol efflux from cholesterol-loaded J774 cells. However, cholesterol associated with TG-enriched HDL was inefficiently esterified by lecithin:cholesterol acyltransferase, and TG-enriched HDLs were poor donors of CE to HepG2 hepatocytes by selective uptake.We conclude that TG-enrichment, in the absence of other significant alterations in lipoprotein composition, is sufficient to alter both cholesterol delivery and removal mechanisms. Some of these abnormalities may contribute to increased coronary disease in hypertriglyceridemia.

It has been shown that estrogens need to be metabolized to their hydrophobic estrogen ester derivatives to act as antioxidants in lipoproteins. Data suggest that 17β-estradiol (E2) becomes esterified in LCAT-induced reactions and the esters are transported from HDL particles to LDL and VLDL particles by a CETP-dependent mechanism. In the present study we have further investigated the regulation of E2 esterification by LCAT and focused on the importance of HDL structure and composition in the esterification process. Isolated LDL, HDL2, HDL3, and reconstituted discoidal HDL (rHDL) were incubated with labeled E2, with and without purified LCAT, at 37°C for 24 h. After purification of the lipoprotein fractions, there was a significant peak of radioactivity representing esterified estradiol attached to HDL3 and rHDL, but HDL2 and LDL contained only trace amounts of labeled estradiol ester. TLC analysis confirmed that the radioactivity migrated in a position corresponding to that of 17β-E2 17-monoester standard. The amount of radioactivity associated with HDL3 and rHDL representing esterified E2 was significantly increased by addition of purified LCAT. However, only limited increases of radioactivity were observed in HDL2 and LDL. In conclusion, HDL subfractions differ in their potential to regulate estradiol esterification by LCAT. —Höckerstedt, A., M. J. Tikkanen, and M. Jauhiainen. LCAT facilitates transacylation of 17β-estradiol in the presence of HDL3 subfraction.

Strain CAST/Ei (CAST) mice exhibit unusually low levels of high density lipoproteins (HDL) as compared with most other strains of mice, including C57BL/6J (B6). This appears to be due in part to a functional deficiency of lecithin:cholesterol acyltransferase (LCAT). LCAT mRNA expression in CAST mice is normal, but the mice exhibit several characteristics consistent with functional deficiency. First, the activity and mass of LCAT in plasma and in HDL of CAST mice were reduced significantly. Second, the HDL of CAST mice were relatively poor in phospholipids and cholesteryl esters, but rich in free cholesterol and apolipoprotein A-I (apoA-I). Third, the adrenals of CAST mice were depleted of cholesteryl esters, a phenotype similar to that observed in LCAT- and acyl-CoA:cholesterol acyltransferase-deficient mice. Fourth, in common with LCAT-deficient mice, CAST mice contained triglyceride-rich lipoproteins with “panhandle”-like protrusions. To examine the genetic bases of these differences, we studied HDL lipid levels in an intercross between strain CAST and the common laboratory strain B6 on a low fat, chow diet as well as a high fat, atherogenic diet. HDL levels exhibited complex inheritance, as 12 quantitative trait loci with significant or suggestive likelihood of observed data scores were identified. Several of the loci occurred over plausible candidate genes and these were investigated. The results indicate that the functional LCAT deficiency is unlikely to be due to variations of the LCAT gene. Our results suggest that novel genes are likely to be important in the control of HDL metabolism, and they provide evidence of genetic factors influencing the interaction of LCAT with HDL.—Mehrabian, M., L. W. Castellani, P-Z. Wen, J. Wong, T. Rithaporn, S. Y. Hama, G. P. Hough, D. Johnson, J. J. Albers, G. A. Mottino, J. S. Frank, M. Navab, A. M. Fogelman, and A. J. Lusis. Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei × C57BL/6J). J. Lipid Res. 2000. 41: 1936–1946.

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 ± 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 ± 4 and 65 ± 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose–response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were “capped” with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB “cap” after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity.—Bielicki, J. K., and T. M. Forte. Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity. J. Lipid Res. 1999. 40: 948–954.

Low levels of high density lipoprotein cholesterol (HDL-C) are associated with increased risk of coronary heart disease and, in the United States, are often associated with hypertriglyceridemia and obesity. In Turkey, low HDL-C levels are highly prevalent, 53% of men and 26% of women having HDL-C levels 50th percentile for men and women in the United States were excluded from the analysis. As no dietary or behavioral factors have been identified in the Turkish population that account for increased hepatic triglyceride lipase activity, the elevation most likely has a genetic basis. —Bersot, T. P., G. L. Vega, S. M. Grundy, K. E. Palaoğlu, P. Atagündüz, S. Özbayrakçi, O. Gökdemir, and R. W. Mahley. Elevated hepatic lipase activity and low levels of high density lipoprotein in a normotriglyceridemic, nonobese Turkish population. J. Lipid Res.40: 432–438.

A molecular model was built for human lecithin:cholesterol acyltransferase (LCAT) based upon the structural homology between this enzyme and lipases (Peelman et al. 1998. Prot. Sci. 7: 585–597). We proposed that LCAT belongs to the α/β hydrolase fold family, and that the central domain of LCAT consists of a mixed seven-stranded β-pleated sheet with four α-helices and loops linking the β-strands. The catalytic triad of LCAT was identified as Asp345 and His377, as well as Ser181. This model is used here for the interpretation of the structural defects linked to the point mutations identified in LCAT, which cause either familial LCAT deficiency (FLD) or fish-eye disease (FED). We show that these mutations occur in separate domains of the 3D structure of the enzyme. Most mutations causing familial LCAT deficiency are either clustered in the vicinity of the catalytic triad or affect conserved structural elements in LCAT. Most mutations causing fish-eye disease are localized on the outer hydrophilic surface of the amphipathic helical segments. These mutations affect only minimally the overall structure of the enzyme, but are likely to impair the interaction of the enzyme with its co-factor and/or substrate.—Peelman, F., J-L. Verschelde, B. Vanloo, C. Ampe, C. Labeur, J. Tavernier, J. Vanderkerckhove, and M. Rosseneu. Effects of natural mutations in lecithin:cholesterol acyltransferase on the enzyme structure and activity. J. Lipid Res. 1999. 40: 59–69.

The effects of polyunsaturated fatty acids (PUFA) on the structure of recombinant high density lipoprotein (rHDL) was investigated using homogeneous particles containing phosphatidylcholine (PC), [3H]cholesterol, and apolipoprotein A-I (apoA-I). The PC component of the rHDL contained sn-1 16:0 and sn-2 18:1 (POPC), 18:2 (PLPC), 20:4 (PAPC), 20:5 n–3 (PEPC), or 22:6 n–3 (PDPC). The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.57–1.70 m) compared to those containing POPC (2.83 m). Intrinsic apoA-I tryptophan fluorescence emission intensity and lifetimes were decreased for rHDL containing long chain PUFA compared to POPC and PLPC rHDL. Monoclonal antibody binding studies demonstrated that apoA-I had decreased immunoreactivity with monoclonal antibodies spanning amino acid residues 115–147 in rHDL containing long chain PUFA. PC lipid fluidity, measured as diphenylhexatriene (DPH) fluorescence polarization, was increased in PUFA rHDL compared to POPC rHDL. There also was a strong correlation between the number of sn-2 double bonds in rHDL and DPH fluorescence lifetime (r2 = 0.89). LCAT reactivity of the homogeneous size rHDL was ordered POPC = PLPC>PAPC> PEPC>PDPC. We conclude that rHDL with long chain PUFA in the sn-2 position of PC contain apoA-I that is less stable and in a different conformation than that in POPC rHDL and have a fatty acyl region that is more fluid and hydrated. The weaker interaction of apoA-I with PC containing PUFA may lead to hypercatabolism of apoA-I in plasma explaining, in part, the decreased plasma HDL and apoA-I concentrations seen with PUFA diets.—Huggins, K. W., L. K. Curtiss, A. K. Gebre, and J. S. Parks. Effect of long chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine on the interaction with recombinant high density lipoprotein apolipoprotein A-I. J. Lipid Res. 1998. 39: 2423–2431.

Familial hypercholesterolemia (FH), a disease caused by a variety of mutations in the low density lipoprotein receptor (LDLr) gene, leads not only to elevated LDL-cholesterol (C) concentrations but to reduced high density lipoprotein (HDL)-C and apolipoprotein (apo) A-I concentrations as well. The reductions in HDL-C and apoA-I are the consequence of the combined metabolic defects of increased apoA-I catabolism and decreased apoA-I synthesis. The present studies were designed to test the hypothesis that overexpression of human lecithin:cholesterol acyltransferase (hLCAT), a pivotal enzyme involved in HDL metabolism, in LDLr defective rabbits would increase HDL-C and apoA-I concentrations. Two groups of hLCAT transgenic rabbits were established: 1) hLCAT+/LDLr heterozygotes (LDLr+/–) and 2) hLCAT+/LDLr homozygotes (LDLr–/–). Data for hLCAT+ rabbits were compared to those of nontransgenic (hLCAT–) rabbits of the same LDLr status. In LDLr+/– rabbits, HDL-C and apoA-I concentrations (mg/dl), respectively, were significantly greater in hLCAT+ (62 ± 8, 59 ± 4) relative to hLCAT–rabbits (21 ± 1, 26 ± 2). This was, likewise, the case when hLCAT+/LDLr–/– (27 ± 2, 19 ± 6) and hLCAT–/LDLr–/– (5 ± 1, 6 ± 2) rabbits were compared. Kinetic experiments demonstrated that the fractional catabolic rate (FCR, d–1) of apoA-I was substantially delayed in hLCAT+ (0.376 ± 0.025) versus hLCAT– (0.588) LDLr+/– rabbits, as well as in hLCAT+ (0.666 ± 0.033) versus hLCAT– (1.194 ± 0.138) LDLr–/– rabbits. ApoA-I production rate (PR, mg·kg·d–1) was greater in both hLCAT+/LDLr+/– (10 ± 2 vs. 6) and hLCAT+/LDLr–/– (9 ± 1 vs. 4 ± 1) rabbits. Significant correlations (P < 0.02) were observed between plasma LCAT activity and HDL-C (r = 0.857), apoA-I FCR (r = –0.774), and apoA-I PR (r = 0.771), while HDL-C correlated with both apoA-I FCR (–0.812) and PR (0.751). In summary, these data indicate that hLCAT overexpression in LDLr defective rabbits increases HDL-C and apoA-I concentrations by both decreasing apoA-I catabolism and increasing apoA-I synthesis, thus correcting the metabolic defects responsible for the hypoalphalipoproteinemia observed in LDLr deficiency.—Brousseau, M. E., J. Wang, S. J. Demosky, Jr., B. L. Vaisman, G. D. Talley, S. Santamarina-Fojo, H. B. Brewer, Jr., and J. M. Hoeg. Correction of hypoalphalipoproteinemia in LDL receptor-deficient rabbits by lecithin:cholesterol acyltransferase.

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 ± 40 μg/ml for women, and 84 ± 49 m/ml for men. Prebeta-1 HDL represented 5.5 ± 3.3% of total apolipoprotein A-I in women, and 7.2 ± 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.—O'Connor, P. M., B. R. Zysow, S. A. Schoenhaus, B. Y. Ishida, S. T. Kunitake, J. M. Naya-Vigne, P. N. Duchateau, R. F. Redberg, S. J. Spencer, S. Mark, M. Mazur, D. C. Heilbron, R. B. Jaffe, M. J. Malloy, and J. P. Kane. Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma, age, and gender.

We report on the effect of human apolipoprotein (apo) A-II transgene expression on atherosclerosis susceptibility in two transgenic lines (25.3 and 11.1) whose plasma human apoA-II concentrations (∼23 and 96 mg/dl, respectively) span the normal range in humans. After 9 months of an atherogenic diet, 25.3 and 11.1 transgenic mice developed aortic atherosclerotic lesions that were ∼1.7- and 7-fold, respectively, more extensive than those of non-transgenic control mice. However, there was no difference in the area of atherosclerosis of transgenic and control mice when fed a regular chow diet. This contrasts with the findings in murine apoA-II transgenic mice and provides evidence of a species-specific characteristic that could be of relevance with respect to the high fat intake diets common in most industrialized countries. A possible mechanism of the pro-atherogenic action of human apoA-II could be the inhibition of reverse cholesterol transport and, in support of this, we observed an impairment of apoA-I-HDL particle inter-conversion in the plasma of 11.1 transgenic mice caused, at least in part, by a marked decrease in the endogenous lecithin:cholesterol acyltransferase activity.—Escolà-Gil, J. C., À. Marzal-Casacuberta, J. Julve-Gil, B. Y. Ishida, J. Ordóñez-Llanos, L. Chan, F. Gonzãlez-Sastre, and F. Blanco-Vaca. Human apolipoprotein A-II is a pro-atherogenic molecule when it is expressed in transgenic mice at a level similar to that in humans: evidence of a potentially relevant species-specific interaction with diet.