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Cholesteryl ester

Cholesteryl esters are water-insoluble formed by the esterification of with long-chain s via an between the cholesterol hydroxyl group and the fatty acid carboxylate, enabling efficient storage and transport of cholesterol in biological systems. Their structure renders them more hydrophobic than free cholesterol, allowing them to form the non-polar core of lipoproteins and cytoplasmic lipid droplets surrounded by a . The formation of cholesteryl esters occurs through enzymatic processes, primarily involving lecithin-cholesterol acyltransferase (LCAT) in plasma, which esterifies about 70% of circulating , and acyl-CoA:cholesterol acyltransferase (ACAT) intracellularly for storage. (CETP) facilitates the exchange of cholesteryl esters between lipoproteins, such as from HDL to LDL and VLDL, playing a key role in cholesterol distribution and . These processes prevent the of excess free by converting it into a neutral, storable form that undergoes a continual cycle of and re-esterification with a typical of around 24 hours in cells. In biological function, cholesteryl esters serve as the primary transport form within lipoproteins like HDL and LDL for reverse transport and delivery to tissues, while intracellularly they accumulate in droplets for long-term storage, particularly in cells like macrophages. They exhibit low solubility in monolayers (approximately 7 wt% or 0.8–8 mol%), adopting conformations such as horseshoe or extended shapes that influence their and mobilization. by enzymes like esterase reverses this process, releasing free for cellular use or incorporation. Dysregulation of cholesteryl ester metabolism contributes to , notably , where excessive accumulation in foam cells forms lipid-laden plaques in arterial walls. Oxidized cholesteryl esters, present in atherosclerotic lesions (up to 23% of cholesteryl linoleate), are biomarkers for , with plasma levels ranging from 3–920 nmol/L and reduced by treatments like . Inherited disorders such as cholesteryl ester storage further highlight their role, involving lysosomal accumulation due to deficient .

Structure and properties

Chemical structure

Cholesteryl esters are esters formed by the esterification of the 3β-hydroxyl group at the position of with the carboxyl group of a , yielding a that is more hydrophobic and less polar than free . The general of cholesteryl esters is \ce{C27H45O-OCOR}, where R denotes the alkyl chain of the ; common examples include palmitate (16:0), oleate (18:1), and linoleate (18:2), which predominate in human plasma cholesteryl esters.90002-X)00538-5/fulltext) The molecular structure features a rigid four-ring cyclopentanoperhydrophenanthrene nucleus derived from , with an eight-carbon isooctyl attached at C17, an linkage at C3 connecting to the , and an acyl chain of variable length typically spanning 14 to 24 carbons.90002-X) Cholesteryl esters exhibit structural variations based on the moiety, such as saturated versus mono-unsaturated chains; saturated esters promote denser molecular packing through linear chain alignment, whereas mono-unsaturated esters, with their characteristic kinks from double bonds (e.g., at Δ⁹ in oleate), reduce packing density and influence phase behavior.90002-X)

Physical properties

Cholesteryl esters are highly hydrophobic molecules, with octanol-water partition coefficients () estimated at around 18 for common variants like cholesteryl palmitate, far exceeding the of approximately 8.7 for free . This enhanced results in virtual insolubility in , typically on the order of 10^{-12} mg/L or less, necessitating their incorporation into carriers for aqueous environments. The structural basis for this hydrophobicity lies in the ester linkage to long-chain fatty acids, which amplifies the nonpolar surface area compared to unesterified . The physical state of cholesteryl esters is heavily influenced by the chain length and saturation, leading to varied s. For instance, cholesteryl palmitate, esterified with a saturated C16 chain, exhibits a of 75–77°C, forming a solid at . In contrast, cholesteryl oleate, with an unsaturated C18:1 chain, melts at 42–48°C, remaining under physiological conditions near 37°C. These transitions reflect the packing of the alkyl chains, where unsaturation disrupts crystalline order and lowers the melting temperature. Cholesteryl esters possess a of approximately 0.93 g/cm³, consistent with their nonpolar, lipid-like composition. They also demonstrate optical activity stemming from the multiple chiral centers in the backbone, yielding specific rotations of -24° (in ) for cholesteryl palmitate and -20° (in THF) for cholesteryl oleate. Furthermore, these compounds readily form crystalline phases, including cholesteric and smectic structures, alongside crystalline and isotropic states, particularly in nonpolar solvents where they can self-assemble into ordered mesophases relevant to their phase behavior.

Biosynthesis

Esterification reaction

The esterification of represents a critical step in , converting free into cholesteryl esters for efficient storage and transport within cells and . This reaction involves the covalent attachment of a fatty acyl chain to the hydroxyl group at the 3β-position of , rendering the molecule more nonpolar and less toxic to cellular membranes. The process is essential in tissues where cholesterol influx exceeds immediate needs, preventing from excess free accumulation. Cholesteryl ester biosynthesis occurs via two primary pathways: the intracellular acyl-CoA:cholesterol acyltransferase (ACAT)-mediated reaction and the plasma lecithin-cholesterol acyltransferase (LCAT)-mediated reaction. The ACAT pathway utilizes as the acyl donor and predominates in cellular storage, while the LCAT pathway transfers the acyl group from (lecithin) and is crucial for maturation in circulation. The biochemical reaction for the ACAT pathway can be summarized by the following equation: \text{Cholesterol} + \text{R-CO-SCoA} \rightarrow \text{R-COO-cholesterol (cholesteryl ester)} + \text{CoA} Here, R denotes the fatty acyl chain, typically 16–18 carbons in length, derived from various fatty acids. This transacylation transfers the acyl group from the thioester bond of acyl-CoA to cholesterol, releasing free coenzyme A (CoASH). The reaction is reversible under in vitro conditions but is strongly favored in the forward direction in vivo, driven by cellular compartmentalization, CoA utilization in other pathways, and the physiological imperative to sequester surplus cholesterol. In contrast, the LCAT pathway involves the transfer of a fatty acyl chain from the sn-2 position of phosphatidylcholine to cholesterol: \text{Cholesterol} + \text{1-acyl-sn-glycero-3-phosphocholine (from phosphatidylcholine)} \rightarrow \text{R-COO-cholesterol} + \text{2-lysophosphatidylcholine} This reaction occurs primarily on high-density lipoprotein (HDL) particles in plasma, where LCAT is activated by apolipoprotein A-I (apoA-I), and favors unsaturated fatty acids like linoleate for esterification. At the molecular level, the ACAT mechanism proceeds through . The 3β-hydroxyl group of is deprotonated by a residue (e.g., His460 in ACAT1) acting as a general base within the enzyme's , generating an ion. This then attacks the electrophilic carbonyl carbon of , forming a tetrahedral that collapses to yield the ester bond and expel the thiolate of . The reaction occurs within the plane of the membrane, where both substrates and the catalyzing enzymes are localized. LCAT employs a similar nucleophilic but uses a to activate the hydroxyl for attack on the sn-2 acyl ester of , producing as a byproduct. LCAT is a soluble secreted by the liver and circulates bound to HDL. The primary substrates for ACAT are free and thioesters. Free is sourced from dietary absorption in intestinal enterocytes, endogenous synthesis via the in hepatocytes, or receptor-mediated uptake from lipoproteins such as LDL in macrophages and other peripheral cells. is generated through the ATP-dependent activation of free fatty acids by acyl-CoA synthetases, providing a diverse pool of acyl chains that influence the composition of resulting cholesteryl esters. For LCAT, substrates include free on nascent HDL and plasma , with activity regulated by HDL maturation state. This esterification predominantly takes place in the of key cell types involved in handling for ACAT: enterocytes for packaging into chylomicrons, hepatocytes for incorporation into very low-density lipoproteins, and macrophages for storage in during formation. LCAT acts extracellularly in . The ER localization for ACAT ensures proximity to synthesis machinery and biogenesis sites, facilitating rapid response to fluctuating levels. Regulation of the esterification reaction balances , with high free levels acutely stimulating activity to promote ester formation and avert disruption. Transcriptionally, the ACAT process is modulated via the regulatory element-binding protein (SREBP) pathway: elevated free suppresses SREBP activation and nuclear translocation, thereby inhibiting expression of ACAT2 (the predominant isoform in liver and intestine) and providing to limit further esterification capacity under chronic excess. LCAT activity is regulated post-translationally by apoA-I activation and inhibited by excess free or . This dual regulation—post-translational activation by substrate availability and transcriptional restraint via SREBP for ACAT, and allosteric activation for LCAT—ensures adaptive control without over-accumulation of esters.

Enzymes involved

The key enzymes responsible for cholesteryl ester synthesis are lecithin-cholesterol acyltransferase (LCAT) in plasma and the acyl-CoA:cholesterol acyltransferase (ACAT) isoforms, ACAT1 and ACAT2, in the membrane. LCAT and ACATs were first identified in the mid-20th century as mediators of esterification, with and characterization occurring in the for ACATs and for LCAT. Crystal structures of ACAT isoforms, solved in the early 2020s using cryo-electron microscopy, reveal a conserved featuring a histidine-serine catalytic dyad essential for acyl transfer, embedded within a bundle of transmembrane helices that segregate the active sites to the cytosolic leaflet. LCAT structures, determined by in the , show a α/β fold with a that opens upon binding on HDL surfaces. LCAT, encoded by the LCAT gene on human , is synthesized in the liver and circulates as a associated with HDL. It plays a central role in reverse cholesterol transport by esterifying free on nascent HDL particles, promoting their maturation into spherical HDL2 and facilitating cholesterol efflux from peripheral tissues. LCAT exhibits a strong preference for polyunsaturated fatty acids such as (18:2), resulting in cholesteryl linoleate as the predominant in plasma HDL. ACAT1, encoded by the SOAT1 on human , is ubiquitously expressed but plays a prominent role in steroidogenic tissues such as the adrenal glands and gonads, where it facilitates storage for , as well as in macrophages to prevent free toxicity. It exhibits a preference for (18:1) as a fatty acyl , contributing to the formation of cholesteryl oleate droplets in lipid-laden cells. In contrast, ACAT2, encoded by the SOAT2 on , is primarily expressed in hepatocytes and enterocytes of the liver and , where it selectively esterifies for incorporation into B-containing lipoproteins like very low-density lipoprotein (VLDL) and chylomicrons, aiding dietary and hepatic export. ACAT2 shows broader substrate specificity toward unsaturated fatty acids, including polyunsaturated ones such as arachidonic (20:4) and eicosapentaenoic (20:5) acids, resulting in cholesteryl esters enriched in these moieties within circulating lipoproteins. Efforts to therapeutically target ACATs for anti-atherosclerosis applications have focused on isoform-selective inhibitors, as ACAT1 inhibition in macrophages may reduce formation while ACAT2 inhibition in the intestine and liver could limit atherogenic production; however, early non-selective inhibitors like avasimibe failed in clinical trials due to adverse effects. Ongoing research explores refined inhibitors to exploit these tissue-specific roles without disrupting essential . LCAT modulation is also under investigation, with genetic deficiencies linking low activity to increased cardiovascular risk.

Biological functions

Role in lipid storage

Cholesteryl esters (CEs) serve as the primary intracellular storage form of in various cell types, particularly where excess must be sequestered to maintain cellular . In non-hepatic cells such as adipocytes, cells (-laden macrophages), and steroidogenic cells, CEs accumulate in the neutral core of cytoplasmic droplets (LDs), which are surrounded by a . This storage mechanism prevents the accumulation of free , which can disrupt membrane integrity and induce at high levels. These in adipocytes, foam cells, and steroidogenic cells enable efficient cholesterol buffering, allowing cells to handle fluctuations in cholesterol influx without altering or function. For instance, during lipid loading in macrophages, the majority of incoming cholesterol can be esterified and stored as CEs within LDs, minimizing and supporting cellular demands. The evolutionary advantage of this system lies in its ability to tightly regulate intracellular cholesterol levels, a critical adaptation for preventing membrane perturbations in diverse physiological contexts. Mobilization of stored CEs occurs through regulated , providing free for or export as needed. LDs are coated with perilipin family proteins, such as and 2, which stabilize the droplets and control access by lipases during this process. This dynamic storage and retrieval system ensures cholesterol availability without compromising cellular viability.

Role in lipoprotein transport

Cholesteryl esters (CEs) serve as a primary core component in major lipoproteins, including (HDL), (LDL), and (VLDL), where they occupy the hydrophobic interior and constitute approximately 15-48% of the particle mass depending on the class. In LDL, CEs comprise 37-48% of the total mass, forming the bulk of the neutral lipid core alongside triglycerides, while in HDL they account for 15-30%, and in VLDL for 12-15%. This core structure allows CEs to solubilize and transport systemically, preventing its precipitation in aqueous . Assembly of CEs into lipoproteins occurs primarily in the intestine and liver. In enterocytes, :cholesterol acyltransferase 2 (ACAT2) esterifies absorbed free , incorporating the resulting CEs into the core of chylomicrons for into the and subsequent entry into circulation. Similarly, in hepatocytes, ACAT2 catalyzes CE formation that is packaged into VLDL particles, enabling hepatic export of to peripheral tissues. For reverse , nascent discoidal HDL particles acquire free from peripheral cells and are matured by lecithin- acyltransferase (LCAT), which esterifies the cholesterol to form CEs that drive the particles into spherical HDL with a stable core. The presence of CEs in the core contributes to their buoyancy and gradient separation during ultracentrifugation, as the low- neutral displace higher- proteins and phospholipids toward the surface. For instance, HDL exhibits a higher CE-to-protein than LDL, resulting in relatively lower despite its smaller size, which facilitates efficient reverse transport. CEs enable the solubilization of otherwise insoluble within amphipathic envelopes, allowing its safe delivery to peripheral tissues for and steroidogenesis while minimizing .

Metabolism

Hydrolysis

The hydrolysis of cholesteryl esters (CE) represents the catabolic reversal of esterification, breaking down stored or internalized CE into free cholesterol and a fatty acid via enzymatic cleavage of the ester bond. This process is essential for liberating cholesterol for cellular use, such as membrane synthesis or efflux from cells like macrophages. The general reaction catalyzed by hydrolases is: \text{Cholesteryl ester} + \text{H}_2\text{O} \rightarrow \text{Cholesterol} + \text{R-COOH} where R-COOH denotes the released chain. In eukaryotic cells, CE hydrolysis occurs primarily through two distinct enzymes: lysosomal acid (LAL) and neutral cholesterol ester 1 (NCEH1), each adapted to specific cellular compartments and physiological contexts. LAL, encoded by the LIPA gene, is the principal lysosomal enzyme responsible for hydrolyzing CE and triglycerides derived from endocytosed lipoproteins, such as (LDL) particles, within the acidic milieu of lysosomes. It exhibits an optimal activity at pH 4.5–5.0, aligning with the lysosomal environment, and plays a critical role in intracellular degradation to prevent toxic accumulation. Mutations in LIPA that severely impair LAL function lead to Wolman disease, a rare autosomal recessive lysosomal storage disorder characterized by massive CE buildup in organs like the liver, , and adrenal glands, often resulting in infantile lethality. In contrast, NCEH1, encoded by the NCEH1 gene (also known as AADACL1 or KIAA1363), functions as a neutral pH-active with an optimum around pH 7.2, localized primarily to the membrane (with its catalytic domain facing the ) and exhibiting some cytosolic association. This enzyme mobilizes CE stored in cytoplasmic droplets, particularly in macrophages and steroidogenic tissues, facilitating the release of free for reverse cholesterol transport or hormonal synthesis. Unlike LAL, NCEH1 operates independently of and is vital for post-lysosomal CE processing, contributing to the dynamic turnover of intracellular stores during . Regulation of CE hydrolysis integrates with broader lipid metabolic pathways, particularly in macrophages where formation is regulated. For instance, γ (PPARγ) and PPARα agonists have been shown to modulate activities, with PPARα directly upregulating neutral CE expression to enhance and cholesterol efflux. Statins, by inhibiting and reducing overall synthesis, indirectly limit substrate availability for CE formation and subsequent hydrolysis, though direct effects on enzymes remain context-dependent. This enzymatic breakdown is integral to mobilizing stored CE for efflux, preventing excessive retention in cells.

Transfer mechanisms

Cholesteryl esters (CEs) are transferred between particles in through non-hydrolytic mechanisms mediated primarily by specialized proteins, enabling the redistribution of neutral lipids without degradation. The (CETP), a synthesized in the liver, facilitates the bidirectional exchange of CEs from (HDL) to (VLDL) and (LDL) in return for triglycerides (TGs) from those apoB-containing lipoproteins. This neutral lipid exchange process contributes to the remodeling of lipoprotein cores, where CEs enrich the core of VLDL and LDL while TGs accumulate in HDL. The CETP , located on 16q13.1, encodes this 493-amino-acid protein, which circulates at concentrations of approximately 1-2 μg/mL in human . CETP operates through a shuttle mechanism, where its tunnel-shaped structure—featuring a hydrophobic binding pocket approximately 60 Å long—binds CEs and TGs, allowing transfer down concentration gradients between particles. The protein's boomerang-like conformation enables it to interact sequentially with donor and acceptor lipoproteins, promoting neutral lipid unloading into the acceptor core via transient pores or direct . This process is inhibited by pharmaceutical agents such as anacetrapib, a potent that binds to the protein's lipid-binding site, blocking CE transfer and thereby elevating HDL cholesterol levels by up to 140% in clinical studies. Polymorphisms in the CETP , such as the TaqIB (rs708272), are associated with reduced CETP activity and higher HDL cholesterol concentrations; for instance, the B2 correlates with a 3.1 mg/dL per-allele increase in HDL-C and a 24% lower risk of coronary events in population studies. CETP was first identified in the early 1980s through assays demonstrating CE transfer activity in lipoprotein-deficient , with key reports from 1980 attributing the phenomenon to a specific factor in and . Its three-dimensional structure was first elucidated in 2010 using , revealing a neutral lipid-binding pocket that accommodates two CE molecules and supports the tunnel-mediated transfer model. In addition to CETP, other proteins contribute to CE movement, albeit through distinct pathways. Phospholipid transfer protein (PLTP), another liver-derived plasma protein with partial sequence homology to CETP, primarily transfers phospholipids but also exhibits minor CE transfer activity, aiding in HDL remodeling and size regulation during postprandial lipolysis. Scavenger receptor class B type I (SR-BI), a cell-surface receptor expressed on hepatocytes and steroidogenic tissues, mediates selective uptake of CEs from HDL into cells without internalization of the entire lipoprotein particle, facilitating reverse cholesterol transport to the liver.

Clinical significance

In cardiovascular disease

Cholesteryl esters (CE) play a central role in the of , a key process in (CVD). In the arterial wall, macrophages take up oxidized (oxLDL), leading to excess free accumulation. This is then esterified by acyl-CoA: acyltransferase 1 (ACAT1) into CE, which forms lipid droplets within the cells, transforming macrophages into —a hallmark of early atherosclerotic lesions. This foam cell formation promotes plaque buildup by contributing to the inflammatory response and deposition in the intima. The (CETP) exacerbates atherogenesis by facilitating the exchange of CE from (HDL) to (LDL) particles, thereby enriching atherogenic LDL with CE while depleting protective HDL. Genetic variants in the CETP gene, such as the TaqIB polymorphism (B2 allele), are associated with reduced CETP activity, higher HDL levels, and a lower of coronary heart disease (CHD), with carriers showing up to a 15% reduction in composite ischemic CVD events compared to non-carriers. This variant highlights CETP's pro-atherogenic influence, as lower CETP function correlates with decreased CVD susceptibility. Therapeutic strategies targeting CE metabolism have focused on CETP and ACAT inhibition, though with mixed outcomes. CETP inhibitors like torcetrapib, tested in the 2006 ILLUMINATE trial, raised HDL but increased CVD events due to off-target effects such as elevated , leading to its discontinuation. Similarly, evacetrapib in the 2015 ACCELERATE trial failed to reduce despite HDL elevation and LDL reduction, underscoring challenges in translating lipid changes to clinical benefit. ACAT inhibitors, such as avasimibe in the 2006 ACTIVATE trial, did not slow coronary progression and potentially promoted it, highlighting risks of disrupting CE storage in macrophages. Epidemiologically, elevated plasma CE levels, particularly in LDL particles, correlate with increased CHD risk; for instance, higher cholesteryl ester transfer activity is linked to a 22% greater incidence of coronary events in cohort studies, independent of traditional risk factors. In the , related lipid profiles influenced by CETP variants further support this association, with dysregulated CE distribution contributing to a 20-30% heightened CVD risk in susceptible populations.

In storage disorders

Cholesteryl ester storage disease (CESD) represents the milder, later-onset form of lysosomal acid lipase (LAL) deficiency, an autosomal recessive disorder caused by pathogenic variants in the LIPA gene that impair the enzyme's ability to hydrolyze cholesteryl esters and triglycerides in lysosomes. Affected individuals typically present in childhood or adulthood with due to lipid-laden macrophages in the liver, elevated serum levels (), and , including low . The estimated of CESD is approximately 1 in 40,000 among individuals of descent, though it is often underdiagnosed due to its variable and nonspecific symptoms. In contrast, Wolman disease is the severe, infantile form of LAL deficiency, manifesting within weeks of birth and leading to rapid progression with massive accumulation of cholesteryl esters in visceral organs such as the adrenals, liver, and . Infants exhibit , , vomiting, diarrhea, and adrenal calcification, with nearly all cases proving fatal by age 1 year due to multiorgan failure. Diagnosis of both CESD and Wolman disease relies on demonstrating deficient enzyme activity through biochemical assays on peripheral blood leukocytes, fibroblasts, or dried blood spots, often complemented by genetic sequencing to identify biallelic LIPA variants. The treatment of LAL deficiency involves enzyme replacement therapy with sebelipase alfa, a recombinant human approved by the U.S. in December 2015 for long-term use in patients of all ages, which reduces hepatic fat content, improves lipid profiles, and enhances growth in affected individuals. Pathophysiologically, the accumulation of cholesteryl esters and triglycerides within lysosomes due to deficiency disrupts lysosomal integrity and cellular , leading to formation and progressive damage primarily in visceral organs like the liver and , rather than vascular tissues. This lysosomal storage impairs function and contributes to and , underscoring the multisystemic impact of unchecked buildup.