Lecithin:cholesterol Acyltransferase Reaction Research Articles

Abstract 1025: Structural Basis Of Apolipoprotein A1 Activation Of Lecithin:Cholesterol Acyl Transferase In High Density Lipoproteins

Lecithin:cholesterol acyl transferase (LCAT) is responsible for the formation of extracellular cholesteryl ester, which is critical for high density lipoprotein (HDL) maturation. It also modulates numerous HDL physiological functions by impacting particle subspeciation patterns. LCAT is activated by apolipoprotein A1 (APOA1) which forms antiparallel dimers around the edge of nascent HDL. Previous work has suggested that the interface of APOA1 helices 4 and 6, when properly aligned, creates two symmetrical sites for productive LCAT binding. Size exclusion chromatography was used to isolate reconstituted HDL discs containing two molecules of APOA1 chemically crosslinked to 2 LCAT molecules. Mass spectrometry using multiple crosslinkers, including a novel application of formaldehyde, indicated that LCAT interacts with both APOA1 helices 4 and 6. Cryo-electron microscopy (cryoEM) was used to determine a 10 Å structure of the complex. Using the HADDOCK suite of molecular docking tools, we developed a structural model of LCAT bound to APOA1 discs that is consistent with the preponderance of data accumulated so far. The model shows that LCAT may bind at the two symmetrical sites in different orientations, possibly reflecting different steps of the LCAT reaction. Work to improve the resolution of the cryoEM datasets is ongoing. Further work, using strategically designed cysteine mutations in APOA1 to lock helices 4 and 6 together, suggests that the helices do not move apart during the LCAT reaction process. Determination of the structural interactions between LCAT and its activator, APOA1, is crucial to understanding HDL maturation, which has significant clinical implications. This work provides the highest resolution structural model available for this important interaction.

Influence of lecithin cholesterol acyltransferase alteration during different pathophysiologic conditions: A 45years bibliometrics analysis.

Background: Lecithin cholesterol acyltransferase (LCAT) is an important enzyme responsible for free cholesterol (FC) esterification, which is critical for high density lipoprotein (HDL) maturation and the completion of the reverse cholesterol transport (RCT) process. Plasma LCAT activity and concentration showed various patterns under different physiological and pathological conditions. Research on LCAT has grown rapidly over the past 50 years, but there are no bibliometric studies summarizing this field as a whole. This study aimed to use the bibliometric analysis to demonstrate the trends in LCAT publications, thus offering a brief perspective with regard to future developments in this field. Methods: We used the Web of Science Core Collection to retrieve LCAT-related studies published from 1975 to 2020. The data were further analyzed in the number of studies, the journal which published the most LCAT-related studies, co-authorship network, co-country network, co-institute network, co-reference and the keywords burst by CiteSpace V 5.7. Results: 2584 publications contained 55,311 references were used to analyzed. The number of included articles fluctuated in each year. We found that Journal of lipid research published the most LCAT-related studies. Among all the authors who work on LCAT, they tend to collaborate with a relatively stable group of collaborators to generate several major authors clusters which Albers, J. published the most studies (n = 53). The United States of America contributed the greatest proportion (n = 1036) of LCAT-related studies. The LCAT-related studies have been focused on the vascular disease, lecithin-cholesterol acyltransferase reaction, phospholipid, cholesterol efflux, chronic kidney disease, milk fever, nephrotic syndrome, platelet-activating factor acetylhydrolase, reconstituted lpa-i, reverse cholesterol transport. Four main research frontiers in terms of burst strength for LCAT-related studies including "transgenic mice", "oxidative stress", "risk", and "cholesterol metabolism "need more attention. Conclusion: This is the first study that demonstrated the trends and future development in LCAT publications. Further studies should focus on the accurate metabolic process of LCAT dependent or independent of RCT using metabolic marker tracking techniques. It was also well worth to further studying the possibility that LCAT may qualify as a biomarker for risk prediction and clinical treatment.

Portulaca oleracea extract increases lecithin:cholesterol acyltransferase and paraoxonase 1 activities and enhances reverse cholesterol transport in streptozotocin-induced diabetic rat

Background: Plant extracts are generally assumed to be more acceptable and less hazardous than synthetic compounds and could be alternative antidiabetic treatments. Portula caoleracea has been used as one of the traditional edible and medicinal plant in Algeria to treat diabetes. The aim of the present study was to determine the effects of lyophilized aqueous extract of Portulaca oleracea on high-density lipoproteins composition, paraoxonase (PON1) and lecithin:cholesterol acyltransferase (LCAT) activities in streptozotocin-induced diabetic rat. Methods: Diabetes was induced intraperitonially by a single injection of streptozotocin (STZ) (60mg/kg bw). Twelve diabetic rats, weighing 263±5g, were divided into two groups fed a casein diet supplemented or not with Portulaca oleracea extract (1g/kg bw), for 4 weeks. Results: At d28, in Portulaca oleracea treated vs untreated diabetic group, glycemia, serum total cholesterol (TC), triacylglycerols (TG) and phospholipids (PL) concentrations were decreased significantly (p<0.05). The hypolipidemic effect induced by Portulaca oleracea extract was due to the reduction of total cholesterol (TC) in LDL-HDL 1 (-51%) and C-HDL 3 (-21%). Portulaca oleracea treatment improved PON1 and LCAT activities by 48%. HDL 3 -UC (acyl group acceptor) and -PL (enzyme substrate) were diminished respectively by 47% and 82%, whereas HDL 2 -CE concentrations (product of LCAT reaction) were increased by 44%. Moreover, HDL-C levels were found to be positively correlated with PON1 activity (r=0.96, p<0.05).Serum, LDL-HDL 1 , HDL 2 and HDL 3 TBARS levels were respectively, 2.9-, 2.6-, 2.4- and 2.8-fold lower in Portulaca oleracea treated than untreated diabetic groups. Conclusion: These findings reflect the potential antihyperglycemic and hypolipidemic of Portulaca oleracea extract, in STZ-induced diabetic rat. Moreover, Portulaca oleracea extract restores PON1 and ameliorates the reverse cholesterol transport (RCT) by enhancing LCAT activity, therefore could prevent many diabetic complications by reducing dyslipidemia and oxidative damage.

Biomarkers associated with high-density lipoproteins in atherosclerotic kidney disease

High-density lipoproteins (HDL) originate as discoidal particles that are rapidly converted by lecithin:cholesterol acyltransferase (LCAT) into the spherical particles that predominate in normal human plasma. Spherical HDL consist of multiple populations of particles that vary widely in size, composition and function. Human population studies have established that high plasma HDL cholesterol levels are associated with a reduced incidence of cardiovascular disease. The mechanistic basis of this relationship is not well understood, but most likely involves a number of the cardioprotective functions of HDL. These include the ability of apolipoprotein (apo) A-I, the main apolipoprotein constituent of HDL, to remove cholesterol from macrophages in the artery wall. HDL also have antioxidant and anti-inflammatory properties that are potentially cardioprotective. Evidence that some of these beneficial properties are compromised in people with diabetes and renal disease is emerging. Persistently elevated plasma glucose levels in people with diabetes and poor glycemic control can lead to irreversible, non-enzymatic glycation of plasma proteins, including apoA-I. Non-enzymatically glycated proteins are also prevalent in people with diabetes and end-stage renal disease who are at high cardiovascular risk. Evidence that non-enzymatically glycated apoA-I inhibits the LCAT reaction and impairs some of the cardioprotective properties of HDL is also emerging. This review is concerned with how non-enzymatic glycation of apoA-I affects the ability of LCAT to convert discoidal HDL into spherical HDL, how it affects cholesterol efflux from macrophages and how it affects the anti-inflammatory and antioxidant properties of HDL.

Ajuga ivaaqueous extract improves reverse cholesterol transport in streptozotocin-induced diabetic rat

The aim of this study was to determine the effects of Ajuga iva aqueous extract on lecithin : cholesterol acyltransferase (LCAT) activity and amount and composition of high-density lipoprotein (HDL)(2) and (HDL)(3), in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar rats by intraperitoneal injection of STZ (60 mg/kg body weight). Diabetic rats (n = 12) were divided into two groups. The diabetic control group (D) received a 20% casein diet and the diabetic treated group received the same diet supplemented with A. iva aqueous extract (0.5 g/100 g diet) (DAi), for 4 weeks. Total cholesterol and HDL(3) -C were respectively decreased by 32% and 55% in the DAi group compared with the D group, whereas HDL(2)-C was increased by 30%. The amounts of HDL(2) and HDL(3), which were the sum of apolipoproteins, unesterified cholesterol (UC), cholesteryl esters (CEs), triacylglycerols (TGs) and phospholipids (PLs), showed no significant difference. A. iva treatment increased LCAT by 33% and its cofactor-activator, apolipoprotein A-I, by 58%. HDL(3)-PL (enzyme substrate) and HDL(3)-UC (acyl group acceptor) were respectively decreased by 70% and 57%, whereas HDL(2)-CE (product of LCAT reaction) was enhanced by 30%. In STZ-induced diabetic rats, A. iva improves reverse cholesterol transport by enhancing LCAT activity, leading to anti-atherogenic effects.

Plasma lecithin:cholesterol acyltransferase and carotid intima-media thickness in European individuals at high cardiovascular risk

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. LCAT is a major factor in HDL remodeling and metabolism, and it has long been believed to play a critical role in macrophage reverse cholesterol transport (RCT). The effect of LCAT on human atherogenesis is still controversial. In the present study, the plasma LCAT concentration was measured in all subjects (n = 540) not on drug treatment at the time of enrollment in the multicenter, longitudinal, observational IMPROVE study. Mean and maximum intima-media thickness (IMT) of the whole carotid tree was measured by B-mode ultrasonography in all subjects. In the entire cohort, LCAT quartiles were not associated with carotid mean and maximum IMT (P for trend 0.95 and 0.18, respectively), also after adjustment for age, gender, HDL-cholesterol (HDL-C), and triglycerides. No association between carotid IMT and LCAT quartiles was observed in men (P=0.30 and P=0.99 for mean and maximum IMT, respectively), whereas carotid IMT increased with LCAT quartiles in women (P for trend 0.14 and 0.019 for mean and maximum IMT, respectively). The present findings support the concept that LCAT is not required for an efficient reverse cholesterol transport and that a low plasma LCAT concentration and activity is not associated with increased atherosclerosis.

Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

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-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg 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-ITg 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-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

The impact of glycation on apolipoprotein A-I structure and its ability to activate lecithin:cholesterol acyltransferase

Hyperglycaemia, one of the main features of diabetes, results in non-enzymatic glycation of plasma proteins, including apolipoprotein A-I (apoA-I), the most abundant apolipoprotein in HDL. The aim of this study was to determine how glycation affects the structure of apoA-I and its ability to activate lecithin:cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport. Discoidal reconstituted HDL (rHDL) containing phosphatidylcholine and apoA-I ([A-I]rHDL) were prepared by the cholate dialysis method and glycated by incubation with methylglyoxal. Glycation of apoA-I was quantified as the reduction in detectable arginine, lysine and tryptophan residues. Methylglyoxal-AGE adduct formation in apoA-I was assessed by immunoblotting. (A-I)rHDL size and surface charge were determined by non-denaturing gradient gel electrophoresis and agarose gel electrophoresis, respectively. The kinetics of the LCAT reaction was investigated by incubating varying concentrations of discoidal (A-I)rHDL with a constant amount of purified enzyme. The conformation of apoA-I was assessed by surface plasmon resonance. Methylglyoxal-mediated modifications of the arginine, lysine and tryptophan residues in lipid-free and lipid-associated apoA-I were time- and concentration-dependent. These modifications altered the conformation of apoA-I in regions critical for LCAT activation and lipid binding. They also decreased (A-I)rHDL size and surface charge. The rate of LCAT-mediated cholesterol esterification in (A-I)rHDL varied according to the level of apoA-I glycation and progressively decreased as the extent of apoA-I glycation increased. It is concluded that glycation of apoA-I may adversely affect reverse cholesterol transport in subjects with diabetes.

Regulation of the Activity and Fatty Acid Specificity of Lecithin-Cholesterol Acyltransferase by Sphingomyelin and Its Metabolites, Ceramide and Ceramide Phosphate

Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the level of inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids on the phospholipase A and cholesterol esterification reactions of the enzyme were similar, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Incorporation of ceramide into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However, these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response.

Measurement of cholesterol bidirectional flux between cells and lipoproteins

We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum.

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.

ESI-MS quantitation of increased sphingomyelin in Niemann-Pick disease type B HDL

HDLs have been proposed to have antiatherogenic properties because of their role in reverse cholesterol transport as lipid acceptors. To elucidate the phospholipid profile of these particles, we used electrospray ionization mass spectrometry to examine the phosphatidylcholine (PC) and sphingomyelin (SM) composition of HDLs purified from plasma and nascently generated in vitro from fibroblasts. We also quantitatively compared the phospholipids present in these lipoproteins between normal and Niemann-Pick disease type B (NPD-B) subjects characterized by sphingomyelinase (SMase) deficiency. We demonstrated that plasma HDLs from NPD-B were significantly enriched in SM by an average of 28%, particularly the palmitoyl SM (with an increase of 95%), which accounted for approximately 25-44% of total SM molecular species. Similarly, we observed an increase of approximately 63% in total SM levels in nascent HDLs prepared from NPD-B fibroblasts. Although PC levels in nascent HDLs were comparable between control and NPD-B cells, there was a 95% increase in total PC levels similar to that of SM in plasma HDLs extracted from NPD-B subjects. These data provide insight into the structure of HDLs and identify potential new roles for SMase in lipoprotein metabolism.

Efeito de tratamento cirúrgico sobre a atividade da enzima hepática lecitina: colesterol aciltransferase (LCAT) na esquistossomose mansônica

A esquistossomose mansônica é uma doença tropical que constitui um importante problema de saúde pública, na Região Nordeste do Brasil, onde é encontrada em alta endemicidade. Essa parasitose tem o fígado como principal alvo de suas lesões histológicas, alterações fisiopatológicas e manifestações clínicas. Estudos anteriores reportam alterações no metabolismo lipídico associadas à forma hepatoesplênica da esquistosomose.Uma das principais alterações consiste na redução da atividade da enzima hepática LCAT, responsável pela esterificação do colesterol no plasma. Neste trabalho, avaliamos a atividade da LCAT no plasma de pacientes portadores da esquistossomose mansônica hepatoesplênica, os quais foram submetidos a esplenectomia e reimplante de parte de tecido do baço. A atividade enzimática da LCAT foi determinada com substrato radioativo. O [14C]colesterol livre e esterificado, formados por ação da LCAT, foram separados por cromatografia em camada delgada e a radioatividade das amostras foi contada em analisador de cintilação líquida. A atividade da LCAT nos pacientes submetidos a esplenectomia e reimplante de tecido do baço apresentou redução de 32 %, em relação ao grupo controle. Contudo, nos portadores da doença que não foram submetidos ao procedimento cirúrgico a redução na atividade da LCAT foi o dobro (64%) da observada em pacientes esplenectomizados e com reimplante de parte do tecido do baço. Esses resultados sugerem haver uma melhora significativa no efeito da forma grave da esquistossomose mansônica sobre a atividade da LCAT.

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I

Advances in understanding of the role of lecithin cholesterol acyltransferase (LCAT) in cholesterol transport

We review the structure and function of lecithin cholesterol acyl transferase (LCAT), the advances in the studies of molecular genetics of LCAT and its deficiency states as well as the developments in assessment of LCAT activity particularly the concept of measurement of fractional esterification rate of plasma cholesterol in the absence of apoB lipoproteins (FERHDL) as an indication of atherogenic risk. We discuss LCAT reaction from two points of view: one that is consistent with the general belief in LCAT antiatherogenic potential and another, namely, a proposed concept of potentially opposing roles of LCAT in normal and dyslipidemic plasmas. While other plasma lipoproteins can (in addition to HDL) provide unesterified cholesterol (UC) for LCAT reaction, HDL may play an unique role in trafficking of newly formed cholesteryl esters (CE) rather than as a primary acceptor of cellular cholesterol. Thus, the plasma HDL, specifically the larger (HDL2b) particles, direct the efflux of most of (LCAT produced) CE to its specific catabolic sites rather than to potentially atherogenic VLDLs and back to LDLs.

Binding Affinity and Reactivity of Lecithin Cholesterol Acyltransferase with Native Lipoproteins

The first step in the reaction of lecithin cholesterol acyltransferase (LCAT) with lipoproteins is the interfacial binding of the enzyme to the lipid surfaces. In this study the equilibrium dissociation constants (Kds) for the interaction of pure human plasma LCAT with LDL, HDL2, HDL3, and a reconstituted discoidal HDL (rHDL) were determined by the activity-inhibition method. In addition, enzyme kinetics were measured with each of the lipoprotein substrates. Based on phospholipid concentrations, the Kdvalues (0.9 × 10−5to 4.6 × 10−5M) increased in the order rHDL = HDL3≤ HDL2< LDL while the relative reactivities (app Vmax/app Km) with LCAT were 100, 16, 1, 6%, respectively, for the different lipoproteins. These quantitative measures were used to predict the distribution of LCAT in plasma and to explain cholesterol esterification when HDL are absent or ineffective as substrates.

Reduced serum lecithin:cholesterol acyltransferase activity and cholesteryl ester concentration in calves experimentally inoculated with Pasteurella haemolytica and bovine herpes virus-1.

Lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for esterification of cholesterol in plasma, is reported to be implicated in the regulation of inflammation in laboratory animals. The purpose of the present study was to elucidate the possible relevance of LCAT in the pathogenesis of calf pneumonia induced by inoculations of Pasteurella haemolytica and bovine herpes virus-1 into the calf lung. Serum LCAT activity was significantly (P < 0.01) reduced in calves inoculated with Pasteurella haemolytica. The concentration of cholesteryl esters (CE), the product of the LCAT reaction, was also decreased in the inoculated group. Decreases in LCAT activity and the CE concentration were similarly observed in calves in which bovine herpes virus-1 was inoculated. In both bacteria- and virus-inoculated calves, CE concentrations in the high-density lipoprotein fractions were distinctly decreased, whereas those in the low-density lipoprotein fractions were practically unaltered. The acute-phase proteins haptoglobin and serum amyloid A were detected in sera from the bacteria- and virus-inoculated calves; however, the two acute-phase proteins were also found in sera from the control calves. These results suggest that decreases in LCAT activity and the CE concentration are involved in the pathogenesis of pneumonia induced by inoculation of calves with Pasteurella haemolytica and bovine herpes virus-1, and also that the change in the LCAT system is more intimately related to the occurrence of calf pneumonia than the induction of acute-phase proteins such as haptoglobin.

Cholesteryl ester transfer protein activity enhances plasma cholesteryl ester formation. Studies in CETP transgenic mice and human genetic CETP deficiency.

The plasma cholesteryl ester transfer protein (CETP) promotes the removal of HDL cholesteryl esters and is thought to stimulate reverse cholesterol transport (RCT). However, mechanisms by which CETP may stimulate RCT are poorly understood. Thus, we examined the relationship between plasma CETP expression and plasma cholesteryl ester formation in CETP transgenic (Tg) mice, hamsters, and human subjects with genetic CETP deficiency. Incubation of CETP Tg mouse plasma showed a 20% to 40% increase in plasma cholesterol esterification rate (CER, P < .05) compared with control mice. Injection of a neutralizing CETP monoclonal antibody (MAb) (TP2) into natural flanking region CETP Tg mice resulted in an increase in plasma free cholesterol (FC) concentration, FC/CE ratio, FC/phosphatidylcholine ratio, and hepatic CETP mRNA. In hamsters, CETP inhibition also resulted in an increase in plasma FC/phosphatidylcholine ratio and increased CETP mRNA in adipose tissue. In humans with two common CETP gene mutations (an intron 14 splicing defect and a D442G missense mutation), mean plasma CERs were 39 and 60, respectively, compared with 89 nmol x mL-1 x h-1 in normal subjects. By contrast, lecithin:cholesterol acyltransferase (LCAT) mass was normal in CETP-deficient subjects. MAb neutralization of CETP activity in incubated human plasma did not alter the LCAT reaction, even after supplementation with discoidal HDL and VLDL. Thus, genetic alterations in CETP levels lead to secondary changes in the plasma LCAT reaction, possibly because of remodeling of HDL by CETP acting in concert with other factors in vivo. In human genetic CETP deficiency, a moderate impairment in the plasma LCAT reaction may contribute to a defect in RCT, providing a potential mechanism to explain the recently observed excess of coronary heart disease in these subjects.

Impaired cholesterol esterification in the plasma in patients with breast cancer.

An important factor which determines the movement of cholesterol in and out of the cells is the free cholesterol (FC)/esterified cholesterol (EC) ratio in the plasma. Although this ratio has been shown to be increased in several types of malignancies in humans as well as experimental animals, it is not known whether such an abnormality is found in breast cancer patients. Furthermore, the reasons for such an increase in cancer patients are unknown. We studied the plasma lipid composition and the activity of lecithin-cholesterol acyltransferase (LCAT), the enzyme responsible for the formation of most of EC in human plasma, in 12 women with breast cancer and 9 age-matched control women. The plasma EC concentration was found to be significantly decreased in cancer patients, whereas the FC concentration was unchanged, leading to increased FC/EC ratios (P < 0.05). The concentration of phosphatidylcholine, the acyl donor in the LCAT reaction, was decreased significantly, whereas all other phospholipids were unaffected. The cholesterol-esterifying activity of LCAT was significantly lower in cancer patients, whether assayed with endogenous substrates (P < 0.05), or with an exogenous substrate (P < 0.01). However, another function of the enzyme, namely the lysolecithin acyltransferase activity, was increased (P < 0.02), indicating that the enzyme concentration in plasma may not be decreased. These results show that the increase in the FC/EC ratio in cancer patients is due to an impaired esterification of cholesterol by plasma LCAT, probably due to an alteration in the composition of substrate lipoproteins, or the presence of an inhibitory factor.