Why esterify cholesterol

Technological advances in mass spectrometry and the development of new anti-OxCE antibodies make it possible to validate the presence and quantify the levels of OxCEs in human atherosclerotic lesions and plasma. The article discusses the prospects of measuring OxCE levels in plasma as a novel biomarker assay to evaluate risk of developing cardiovascular disease and efficacy of treatment.

Cholesterol esterification is a mechanism the body uses to store and transfer cholesterol, while at the same time to avoid cellular toxicity of the excess of unesterified often called free cholesterol. However, oxidation of a cholesteryl ester CE drastically changes the part CE plays, from a subdued supporting actor to a contender for the leading role. The script, in other words, the specific physiological or pathological context, defines if oxidized CE OxCE plays a villain or the hero.

Unesterified cholesterol is an essential component of cellular membranes, where it plays both structural and signaling roles, the latter via regulation of lipid rafts and binding to many transmembrane proteins.

In the nervous system, cholesterol is a major component of myelinated sheath of many nerve fibers. Cholesterol is also a precursor for biosynthesis of steroid hormones and bile acids.

Thus, no wonder that cholesterol homeostasis is under tight control to ensure proper cellular and systemic functions. Dysregulation of cholesterol metabolism underlies many pathologies, from cardiovascular disease CVD to neurodegenerative disorders to cancer 1 , 2.

In homeostatic state, cellular cholesterol content is tightly controlled by balancing de novo synthesis, uptake of lipoproteins, export to extracellular milieu, and storage 2 — 4. The strategy for storage and transport of amphipathic cholesterol molecules is their esterification to fatty acids and tight packaging of the resulting hydrophobic CEs in the core of intracellular lipid droplets or circulating lipoproteins Figure 1 —Transport and Storage.

This diagram illustrates different biological processes that involve CEs, using examples described in text, and potential biomarker applications of detecting OxCEs in plasma and atherosclerotic plaques.

Transport: Cholesteryl esters CEs together with triglycerides TGs populate the hydrophobic core of circulating lipoproteins, serving to deliver cholesterol and fatty acids to organs. Shown are representative structures from top to bottom of a TG, a CE with saturated fatty acyl [cholesteryl palmitate], and a CE with polyunsaturated fatty acyl PUFA [cholesteryl arachidonate], the latter is susceptible to oxidation.

Storage: Intracellular lipid droplets predominantly store either CEs, like in macrophage foam cells in atherosclerotic lesions, or TGs, like in adipocytes. Hydroperoxide, endoperoxide and aldehyde groups in OxCEs are reactive and can covalently modify proteins.

OxCEs also activate endothelial cells, but their effects on vascular smooth muscle cells have not been studied. In one example, an immunoassay measuring levels of OxCE-apoA lipoproteins detects reduced levels of this potential biomarker in subjects after treatment with atorvastatin compared to placebo.

The artwork in this figure uses panels originally published in the Journal of Lipid Research. Gonen et al. A monoclonal antibody to assess oxidized cholesteryl esters associated with apoA-I and apoB lipoproteins in human plasma. J Lipid Res ; Inside the cells, after a threshold level in cellular cholesterol mass has been reached, excess cholesterol is esterified in the ER by the enzyme acyl CoA cholesterol acyltransferase ACAT , and the newly synthesized CEs are stored in lipid droplets.

Alternatively, lipid droplets are packaged into autophagosomes and fuse with lysosomes, where the CE is hydrolyzed by lysosomal acid lipase LAL , generating unesterified cholesterol for delivery to cellular membranes or for export 5.

Cellular CEs undergo a continual cycle of hydrolysis and re-esterification with a half-life of about 24 h 6 , 7. In circulation, lower density lipoproteins transport CEs from digestive organs to tissues, and high-density lipoprotein HDL returns excess cholesterol in the form of CE back to the liver.

LDL is internalized by many cell types and thus delivers CEs to tissues. Serving the opposite function, HDL gathers excess of unesterified cholesterol from extrahepatic tissues in order to move it back to the liver. The HDL-associated enzyme lecithin-cholesterol acyltransferase LCAT catalyzes the esterification of free cholesterol with a fatty acyl transferred from the sn-2 position of phosphatidylcholine PC , resulting in the formation of a CE.

This abridged description of CE metabolism illustrates complex pathways involved in keeping CE locked away in hydrophobic cores of lipid droplets and lipoproteins for storage and safe passage through the body—that is until CE undergoes oxidation and becomes biologically active.

The PUFAs are more susceptible to oxidation than cholesterol due to the presence of a weaker C-H bond at the bis-allylic position and will therefore be oxidized preferentially The hydrogen atoms are easily abstracted from the bis-allylic positions of PUFAs to form a lipid radical - the first intermediate of enzymatic or non-enzymatic lipid peroxidation 13 , This article will be largely focused on the OxCEs with oxidized acyl chain and non-oxidized sterol, with a brief discussion of oxysteryl-containing OxCEs.

It is unknown if any of lipid droplet-associated proteins can mediate a similar CE exchange and if CE oxidation can occur on the surface of intracellular lipid droplets.

Among these polyoxygenated CE products, molecules with a bicyclic endoperoxide group 18 — 20 , such as cholesteryl 9,11 -epidioxyhydroperoxy- 5 Z ,13 E -prostadienoate abbreviated as BEP-CE for the presence of bicyclic endoperoxide and hydroperoxide groups , have biological activities and can covalently modify proteins, including apolipoproteins, as discussed below.

OxCEs can also decompose to produce highly reactive end products, like malondialdehyde MDA or 4-hydroxynonenal 4-HNE , which in turn covalently modify proteins and phosphatidylethanolamines 21 , These posttranslational modifications profoundly affect protein function. In addition, intracellular OxCE hydrolysis and subsequent re-esterification of an oxidized fatty acyl chain can produce oxidized PL OxPL in the cell Oxysterols, derived from either enzymatic or non-enzymatic oxidation of cholesterol, are bioactive and play important regulatory roles 23 , Similar to esterification of cholesterol, esterification of oxysterols is mediated by ACAT in cells and LCAT in plasma, as well as by lysosomal phospholipase A2 25 , Transport of OxCEs in circulation and their uptake by cells occur via the same pathways that traffic non-oxidized CEs.

Oxidized cholesteryl linoleate in which the fatty acyl moiety is oxidized to a hydroperoxide moves readily from HDL to hepatoma cells in serum-free medium 35 , and the rate of SR-BI-mediated OxCE uptake by cells is approximately 9 times faster than that of non-oxidized CE and at least 40 times faster than the uptake of a whole HDL lipoprotein 36 , Because of the presence of hydrophilic, oxygen-containing groups in OxCE molecules, they become amphipathic and more mobile and presumably less confined to hydrophobic cores of lipoproteins or lipid droplets.

Unmodified cholesterol is a major regulatory molecule for many proteins. However, we have not seen direct experimental or modeling comparison of cholesterol and long-chain CE or OxCE binding to these domains. And we are unaware of studies of interaction between transmembrane proteins, like GPCRs, with OxCEs, which are bifunctional—carrying both unmodified cholesteryl and fatty acyl oxidation moieties. In addition to transmembrane cholesterol-binding proteins, there are non-membrane proteins that have hydrophobic pockets where cholesterol docks, CETP and Niemann-Pick disease, type C2 protein NPC2 being the most characterized proteins in this class.

Another cholesterol-binding protein is MD-2 Fatty acyl chains of LPS dock into the hydrophobic pocket, and negatively charged phosphate groups of LPS bind positively charged residues at the pocket opening. Likewise, in the molecule of cholesterol, a hydrocarbon chain together with the steroid form an elongated hydrophobic structure, which docks in the hydrophobic pocket of MD-2, and a hydroxyl group linked to the other side of the steroid stabilizes cholesterol at the positively charged entrance to the pocket.

Test-tube experiments confirm that MD-2 binds cholesterol Furthermore, cholesterol is found associated with the MD-2 immunoprecipitated from human plasma or from mouse atherosclerotic lesions It is unlikely that unesterified cholesterol binding to MD-2 activates TLR4 because there is no moiety in the MDbound cholesterol that would interact with TLR4, however, such a moiety is present in the cholesteryl esterified to a fatty acyl—CE.

The hypothesis that an oxygenated fatty acyl chain in OxCE provides additional interaction surfaces, which, in combination with cholesteryl anchoring in the MD-2 hydrophobic pocket, provide sufficient interfaces for OxCE-induced MDTLR4 binding, remains untested. Remarkably, in in vitro experiments, mmLDL and low-dose LPS, imitating subclinical endotoxemia observed in patients with the metabolic syndrome, synergize to produce higher levels of inflammatory cytokines.

Although published data point to pro-inflammatory effects of OxCE, a more extensive literature on biological effects of OxPL describes both pro-and anti-inflammatory effects depending on the disease or pathological condition context Thus, we cannot exclude the possibility of context-dependent, anti-inflammatory effects of OxCE, but this requires further research. In contrast to the biological activity of fatty acyl-oxidized OxCE, esterification of oxysterols largely serves to curtail their biological activity However, in neurons, ACAT-mediated esterification of 24 S -hydroxycholesterol results in the formation of atypical lipid droplets and neurotoxicity These findings suggest cell type and context dependent effects of esterification of oxysterols.

It is also possible that in the experimental systems employed in the above experiments, cholesteryl HPODE underwent further oxidative modifications, resulting in more complex products, which in turn evoked biological activity different from that of an initial hydroperoxide.

How relevant is the biological activity of OxCE to the pathogenesis of atherosclerosis? In the absence of similar work targeting OxCE, we can only hypothesize that the substantial presence of OxCE in atherosclerotic lesions may have an atherogenic effect.

BEP-CE has been detected in human atherosclerotic lesions as well The studies cited in the previous paragraph identified free lipid OxCEs. It is technically challenging to construct a mass spectrometry method that would detect covalent OxCE adducts to proteins. However, these new epitopes can be detected with antibodies raised against the OxCE moiety independent of a protein, which has been covalently modified by this OxCE. For example, a monoclonal antibody raised against proteins modified with cholesteryl 9-ON has been shown to stain atherosclerotic lesions Studies in our lab have produced a new monoclonal antibody that recognizes an OxCE epitope on modified proteins.

The AG23 immunoreactivity was abundant in human carotid endarterectomy specimens, demonstrating the presence of OxCE epitopes in atherosclerotic lesions Figure 1 —Biomarkers and suggesting relevance of OxCE to the pathogenesis of human CVD Plasma levels of CE hydroperoxides have been significantly increased on day 1 and peaked at day 5 after subarachnoid hemorrhage, returning to normal levels on days 7 and 9 This temporal sequence correlates well with the known time course of cerebral vasospasm, which typically has its onset between 5 and 7 days after subarachnoid hemorrhage.

These results suggest release of OxCEs from the raptured atherosclerotic plaque This assay measures levels of lipoproteins that have at least one OxCE epitope.

Measuring OxCE-apoB and OxCE-apoA in plasma samples from PROXI, a randomized parallel-arm double-blind placebo-controlled trial in which human subjects received placebo or a statin treatment for 16 weeks, we demonstrated that the OxCE-apoA levels were significantly lower in subjects treated with atorvastatin than in the placebo group, independent of the apoA-I levels Figure 1 —Biomarkers Biological activity and biomarker potential of OxCEs remain understudied.

However, there exist mechanisms for oxidative modification of CEs with polyunsaturated fatty acyls, producing a multitude of OxCEs, which exhibit biological activity as free lipid and can covalently modify proteins.

In this article, we reviewed how cholesterol binding to MD-2 makes OxCE an agonist for TLR4, resulting in inflammatory and lipid accumulation responses in macrophages. More work is also needed to understand biological effects of esterified oxysterols, which seem to be tissue and pathology context dependent.

The spectrum of proteins in plasma and in tissues that have OxCE covalent adducts remains unexplored, as remain poorly understood determinants of the OxCE specificity in covalent modification of proteins. The development of new antibodies that recognize OxCE-protein covalent complexes independent of a protein carrier will be instrumental in answering these questions.

However, there remains a challenge of characterization of the exact chemical structure of OxCE-protein covalent adducts; where mass spectrometry methods are insufficient, co-crystallization of antibodies with their OxCE antigens may become a productive approach. Initial studies using OxCE-specific antibodies, as illustrated in this article, are promising but examination of larger cohorts of subjects with CVD and possibly other conditions are needed to fully evaluate the diagnostic and prognostic potential of OxCE as a new biomarker.

AG and YM wrote this manuscript. All authors contributed to the article and approved the submitted version. AG and YM are inventors listed on patents and patent applications related to the topic of this article. The terms of this arrangement have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies.

Mechanisms and regulation of cholesterol homeostasis. Nat Rev Mol Cell Biol — Tabas I. Cholesterol in health and disease. J Clin Invest — A receptor-mediated pathway for cholesterol homeostasis.

Science — Molecular Pathways Underlying Cholesterol Homeostasis. Nutrients Front Physiol The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters.

J Biol Chem — PubMed Abstract Google Scholar. For this purpose prothrombin time, cholinesterase and bilirubin are available. These parameters reflect main aspects of liver function. Prothrombin time respectively INR reflect mainly the capacity of the liver synthesis whereas the concentration of bilirubin is a marker for liver metabolism and the excretion of bile.

A well evaluated and widely used prognostic tool for the prediction of three-month mortality is the model of end-stage liver disease MELD score which is also used for organ allocation for orthotropic liver transplantation in many countries worldwide [ 2 — 4 ].

Most importantly, the INR is changed by anticoagulants and substitution of coagulation factors [ 8 — 10 ]. It was primarily established to monitor the efficacy of the antithrombotic therapy of patients treated with vitamin k antagonists. The underlying prothrombin assays asses the global coagulation function and were not standardized for the prediction of prognosis in patients with end-stage liver disease [ 11 , 12 ].

Importantly, the composition of coagulation factors in patients with end-stage liver disease differs significantly compared to patients treated with vitamin k antagonists and even in well standardized patients with oral anticoagulation these functional tests show a substantial inter-assay variability [ 8 , 12 — 14 ].

Aim of this study was to investigate an alternative plasma biomarker to evaluate the liver function and mortality risk in patients with end-stage liver disease. We identified the cholesterol esterification CE fraction in blood which is very closely related to the liver function as a biomarker with potential for a translation in routine diagnostics. Under physiological conditions, cholesterol esterification is under tight control as it is essential for cellular membrane function.

The most important regulatory enzyme for this regulation in blood is the lecithin-cholesterol acyltransferase LCAT which is produced in the liver. In fact, decreased cholesterol esterification fraction in context of liver cirrhosis has already been described in older literature [ 15 , 16 ].

However, the CE fraction has not been systematically evaluated for short-term mortality prediction in patients with end-stage liver disease to date. To our knowledge, since more than 25 years no clinical studies have been published using the cholesterol esterification fraction to monitor liver function. In this study we analyze the plasma cholesterol esterification fraction as a biomarker for liver synthesis in the context of mortality and in comparison to INR.

We enrolled consecutively patients on the liver transplantation list or in evaluation for orthotopic liver transplantation at the University Hospital Leipzig, Germany. The patients required liver transplantation for different indications Table 1.

No additional samples for the study were taken. All measurements were performed in the residual material. After anonymization of the patients the data from the laboratory information system was analyzed retrospectively. The institutional review board of the University of Leipzig approved the study design and confirmed that an additional patient consent was not required for the measurements and the data analysis Institutional Review Board registration number: ff.

Medical records of all patients were retrospectively analyzed to identify potential drug interferences. One patient was excluded from further analysis, due to the intake of rivaroxaban, a new oral anticoagulation drug and another patient was lost in follow up after days, due to a change of the attending transplant center.

This patient was censored at this date for the analysis of one-year mortality. All blood samples were taken for regular clinical laboratory diagnostics and for reporting to the European transplantation organization Eurotransplant.

Creatinine was measured using the enzymatic assay creatinine Plus Ver. Cholesterol esterification fraction was defined as the quotient between esterified cholesterol and total cholesterol. Total Cholesterol and cholesterol ester were analyzed in residual material of citrate plasma.

For all results from citrate plasma, a dilution factor of 1. If residual citrate plasma was unavailable, serum material was used for the analysis 53 cases. The median difference between citrate plasma and serum was 0. Following centrifugation for 10 min at Maximum serum creatinine level was set to 4. The results of measured parameters were analyzed using Excel We considered three-month and one-year survival as primary endpoint, where liver transplant is counted as censoring. Predictive power of parameters on survival where analyzed via Cox-Regression.

The predictive power for three-month and one-year mortality was analyzed for uncensored patients and compared using areas under the curve of the Receiver operating characteristic AUROC.

One hundred forty-two patients suffering from end-stage liver disease were included in the study. The follow-up time was one year. The intake of an oral factor Xa inhibitor Rivaroxaban was identified for one patient. Due to the impact on the INR, this patient was excluded from further analysis. See supplementary material for the quantitative results of other investigated parameters in the context of mortality, sex and age Additional file 1 : Tables S1—S4. Patients were divided in tertiles according to their cholesterol esterification rate.

Next, we analyzed the association of cholesterol esterification fraction with one-year mortality rate Fig. The same approach was used for the association of INR tertiles with mortality Fig. Kaplan-Meier survival analysis for tertiles according to cholesterol esterification and INR: Patients were divided in tertiles according to their a cholesterol esterification fraction and b INR. While for CE all risk groups differ significantly Table 2 , the contrast of the highest INR groups is not significant logrank test.

Censoring is indicated with vertical bars within the curves. None of the patients of the other tertiles had died after three months Table 2. After one year, Table 2 summarizes the mortality after three months and one year according to the CE- and INR tertiles. ROC analysis of single biomarkers for predicting three-month a and one-year b mortality during follow up n. In our work, we identified the plasma cholesterol esterification fraction as a promising biomarker for the prediction of mortality in patients with end-stage liver disease.

Cholesterol esterification fraction was significantly superior to INR in the prediction of three month mortality. There are several explanations for the low cholesterol esterification fraction in end-stage liver disease patients. The cholesterol esterification fraction in plasma depends mainly on the activity of the LCAT lecithin-cholesterol acyltransferase [ 19 ], a genetically highly conserved enzyme.

LCAT is produced by the liver and secreted into the blood. The plasma concentration in persons with normal liver function show only minor variation and the half-life in plasma have been estimated to be 4—5 days [ 20 ]. LCAT has been discovered in [ 21 ] and plays an important role for the reverse cholesterol transport [ 22 ].

The LCAT plasma concentration may also be an interesting biomarker for liver function, but the measurement requires an immunological assay which is expensive, difficult to standardize and the result does not reflect the enzyme activity. In the reaction of cholesterol esterification the abnormal lipoprotein X LP-X [ 24 ] has been described also. LP-X particles contain almost only unesterified cholesterol and appear typically in patients with obstructive biliary diseases and in patients with LCAT-deficiency.

But so far, a quantification of LP-X for clinical routine is not available and the clinical relevance remains unclear [ 25 ]. In terms of methodical validity, the calculation of the esterification fraction from the results of plasma cholesterol ester and total cholesterol could be advantageous.

The implementation of the quotient eliminates interferences with the complex matrix of samples from end-stage liver disease patients. In clinical routine cholesterol is measured on high throughput analyzers with well standardized enzymatic assays. In these assays all cholesterol esters are first hydrolyzed to free cholesterol by cholesterol ester hydrolase.

The free cholesterol is then oxidized by cholesterol oxidase resulting in a chromogen which can be measured photometrically [ 26 ]. The same application without cholesterol ester hydrolase could be used in clinical routine measuring the content of free cholesterol which allows the calculation of the cholesterol ester fraction.

Plasma cholesterol esterification fraction has some important advantages compared to INR as a biomarker for liver disease. Importantly, in contrast to INR cholesterol esterification fraction is not affected by anticoagulants.

The patient who was excluded from further analysis in our study received an anticoagulation treatment with the direct oral factor Xa inhibitor rivaroxaban which is known to affect the INR. In contrast, the cholesterol esterification rate was almost stable See Additional file 1 for details. Prothrombin time as well as other global coagulation tests is difficult to standardize.

These assays are based on the detection of the coagulation time after activation with different thromboplastines. The characteristics of theses thromboplastines differ significantly between available assays.

However, for this standardization, exclusively samples of patients treated with vitamin k antagonists were included as monitoring of this treatment was the intention for implementation of INR. Patients taking vitamin k antagonists compared to patients suffering from liver disease show a significantly different composition of coagulation factors.

Factor V, a central element of the coagulation system, is not influenced by taking vitamin k antagonists, but in patients with liver cirrhosis, it is decreased in accordance with the stage of the liver disease [ 27 ].

Consequently, the results of different assays in patients with end-stage liver disease vary significantly [ 28 , 29 ]. A possible recalibration of the ISI which could eliminate some of the limitations for patients with end-stage liver disease is sophisticated and has not been implemented in clinical routine [ 30 — 33 ]. Until now, the effect of antidyslipidemic drugs on the cholesterol esterification fraction has not been investigated in patients with end-stage liver disease.

We analyzed the cholesterol esterification fraction in the large cohort of patients with coronary artery disease in the Leipzig LIFE Heart Study [ 35 ] and compared patients under statins with patients without statins. The cholesterol esterification fraction did not differ relevantly between these two groups CE rate with statins There are some limitations of our study. The most important one is the limited number of patients and the heterogeneous causes for end-stage liver disease.

Therefore, we were not able to analyze subgroups of patients in this study. If the mortality prediction is also valid for patients with hepatocellular carcinoma cannot be answered yet and will be an important question to answer in future studies.

We currently develop and evaluate an assay for quantification of free cholesterol on a high-throughput clinical chemistry analyzer system which allows, in utilization of a total cholesterol assay, the calculation of cholesterol esterification fraction. Based on this test, we plan a multicenter study to prove the diagnostic value of this rediscovered biomarker for liver function. Further research is also needed to study the metabolism of involved lipoproteins in more detail.

Despite the small sample size, a benefit using the cholesterol ester fraction to estimate the liver function and to predict mortality risk in patients with livers disease in comparison with INR is plausible.

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