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This study is one of the first to focus on the unexpected drug leakage from discoidal recombinant high-density lipoproteins (d-rHDLs) as a consequence of remodeling process, mainly associated with lecithin-cholesterol acyltransferase (LCAT) during their metabolic process. Here, a newly monocholesterylsuccinate (CHS) modified paclitaxel-loaded d-rHDLs (cP-d-rHDLs) were constructed successfully through structural modification, thus aiming to improve the performance of d-rHDLs. And next their in vitro physiochemical properties and pharmacokinetics in Sprague–Dawley rats were elaborately investigated. Collectively our studies demonstrated that cP-d-rHDLs, whose remodeling behaviors were restrained effectively after structural modification, exhibited more excellent and promising properties as novel delivery vehicles for anti-cancer agents.

PubMed: 25572761, Objectives: Diabetes mellitus (DM) is a chronic disease and one of the most important health problems. Several factors may be responsible for the complications of diabetes mellitus including alterations in the activities of sodium-potassium adenosine triphosphatase (Na+/K+ ATPase) and lecithin:cholesterol acyltransferase (LCAT) and also levels of nitric oxide (NO). We have investigated the effects of alterations in serum NO levels on activities of erythrocyte membran Na/K ATPase and serum LCAT enzymes. Materials and methods: The experiments were performed on male rats divided into four groups: group 1, control (standart diet); group 2, diabetic control (single dose of 65 mg/kg of streptozotocin (STZ), i.p); group 3, STZ + insulin (8 IU/kg/day s.c.); group 4 (STZ + l-NAME 5 mg/kg/day orally). Result: Streptozotocin-induced diabetic rats, showed a significant increase in blood glucose and serum cholesterol (C) and triglyceride (TG). Compared to the control group with diabetic group plasma LCAT concentrations and erythrocyte membrane Na +/K+ ATPase were found to be decreased. Activities of Na+/K+ ATPase and serum NO level were decreased with the administration of l-NAME. We observed that insulin was ameliorated in all parameters. Conclusions: Serum NO levels is related to erythrocyte membrane Na+/K+ ATPase activity. But serum NO levels did not affect the plasma LCAT activity and serum lipid profiles

In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preβ1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL.

HMG-CoA reductase, cholesterol 7α-hydroxylase, LCAT, ACAT, LDL receptor, and SRB-1 in hereditary analbuminemia. Background Hereditary analbuminemia is associated with hypercholesterolemia, which has been shown to be primarily caused by increased extrahepatic production of cholesterol. Nagase rats with hereditary analbuminemia (NAR) have been used as a model to dissect the effect of primary hypoalbuminemia from that caused by proteinuria in nephrotic syndrome. The present study was undertaken to explore the effect of hereditary analbuminemia on protein expression of the key factors involved in cholesterol metabolism. Methods Hepatic tissue protein abundance of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase (a rate-limiting enzyme in cholesterol catabolism), low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor (SRB-1), acyl-coA cholesterol acyltransferase-2 (ACAT-2), and plasma concentration of lecithin cholesterol acyltransferase (LCAT), as well as HMG-CoA reductase, ACAT, and LCAT activities were determined in fasting male NAR and Sprague-Dawley control rats. Results The NAR group exhibited significant up-regulation of HMG-CoA reductase protein abundance but normal HMG-CoA reductase enzymatic activity. This was coupled with a significant up-regulation of cholesterol 7α-hydroxylase and a mild up-regulation of ACAT protein abundance and activity. However, hepatic LDL receptor and HDL receptor and plasma LCAT protein concentration and activity were normal in NAR. Conclusion Hypercholesterolemia in NAR is associated with elevated hepatic HMG-CoA reductase protein abundance, but normal HMG-CoA reductase activity. These findings point to post-translational regulation of this enzyme and favor an extrahepatic origin of hypercholesterolemia in NAR. The observed up-regulation of cholesterol 7α-hydroxylase represents a compensatory response to the associated hypercholesterolemia. Unlike nephrotic syndrome, which causes severe LDL receptor, HDL receptor, and LCAT deficiencies, hereditary analbuminemia does not affect these proteins.

We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT. These are mouse anti-human LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486, which react with the peptides corresponding to human LCAT amino acid residues R159-E179, M258-S273, S274-S294, D352-S376, and N415-E440, respectively. We also successfully used two of these antibodies to develop an ELISA, which uses a solid phase monoclonal antibody, 36486, that reacts with the C-terminus of LCAT, and a detection monoclonal antibody, 36487, that reacts with an epitope located in the center of the LCAT primary structure. We observed a significant positive correlation between the values of LCAT protein determined with ELISA and LCAT activity determined with liposome substrate (r = 0.871, P < 0.001) or the endogenous self-substrate method (r = 0.864, P < 0.001), and we obtained inter- and intra-assay coefficients of variation less than 6.1%, minimum detection limit of 0.1 μg/ml. Highly specific monoclonal antibodies will be useful in the study of the molecular pathology of LCAT. Therefore, this precise and sensitive LCAT assay will help clarify the role of this enzyme in the metabolism of HDLs, and can be used for diagnostic purposes in investigating liver function. We obtained five monoclonal antibodies that recognized different epitopes on LCAT and developed a sandwich-type ELISA. Highly specific monoclonal antibodies provide a sensitive and specific analytical system for measurements of LCAT protein.—Kobori, K., K. Saito, S. Ito, K. Kotani, M. Manabe, and T. Kanno. A new enzyme-linked immunosorbent assay with two monoclonal antibodies to specific epitopes measures human lecithin-cholesterol acyltransferase.

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545–552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2 (Ac-18A-NH2 or 2F) were: 3F3(Ac-F318A-NH2), 3F14(Ac-F1418A-NH2), 4F(Ac-F3,1418A-NH2), 5F(Ac-F11,14,17 18A-NH2), 6F(Ac-F10,11,14,17 18A-NH2), and 7F(Ac-F3,10,11,14,17 18A-NH2). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity.These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.—Datta, G., M. Chaddha, S. Hama, M. Navab, A. M. Fogelman, D. W. Garber, V. K. Mishra, R. M. Epand, R. F. Epand, S. Lund-Katz, M. C. Phillips, J. P. Segrest, and G. M. Anantharamaiah. Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide. J. Lipid Res. 2001. 42: 1096–1104.

Lecithin:cholesteryl acyl transferase (LCAT), cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP), and lipoprotein lipases are involved in high density lipoprotein (HDL) metabolism. We evaluated the influence of insulin sensitivity and of the TaqIB CETP gene polymorphism (B1B2) on plasma LCAT, CETP, and PLTP activities (measured with exogenous substrates) and their responses to hyperinsulinemia. Thirty-two non-diabetic men without hyperlipidemia were divided in quartiles of high (Q1) to low (Q4) insulin sensitivity. Plasma total cholesterol, very low + low density lipoprotein cholesterol, triglycerides, and apolipoprotein (apo) B were higher in Q4 compared to Q1 (P < 0.05 for all), whereas HDL cholesterol and apoA-I were lowest in Q4 (P < 0.05 for both). Plasma LCAT activity was higher in Q4 than in Q1 (P < 0.05) and PLTP activity was higher in Q4 than in Q2 (P < 0.05). Insulin sensitivity did not influence plasma CETP activity. Postheparin plasma lipoprotein lipase activity was highest and hepatic lipase activity was lowest in Q1. Insulin infusion decreased PLTP activity (P < 0.05), irrespective of the degree of insulin sensitivity. The CETP genotype exerted no consistent effects on baseline plasma lipoproteins and LCAT, CETP, and PLTP activities. The decrease in plasma PLTP activity after insulin was larger in B1B1 than in B2B2 homozygotes (P < 0.05). These data suggest that insulin sensitivity influences plasma LCAT, PLTP, lipoprotein lipase, and hepatic lipase activities in men. As PLTP, LCAT, and hepatic lipase may enhance reverse cholesterol transport, it is tempting to speculate that high levels of these factors in association with insulin resistance could be involved in an antiatherogenic mechanism. A possible relationship between the CETP genotype and PLTP lowering by insulin warrants further study.—Riemens, S. C., A. Van Tol, B. K. Stulp, and R. P. F. Dullaart. Influence of insulin sensitivity and the TaqIB cholesteryl ester transfer protein gene polymorphism on plasma lecithin:cholesterol acyltransferase and lipid transfer protein activities and their response to hyperinsulinemia in non-diabetic men. J. Lipid Res. 1999. 40: 1467–1474.

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn -2 fatty acid of phospholipid to cholesterol. To facilitate the isola- tion of large quantities of LCAT and to assist in future structure-function studies, LCAT containing a carboxy- terminal histidine-tag (H6) was expressed in Chinese ham- ster ovary cells (CHO). A high level of CHO-hLCATH6 ex- pression ( < 15 mg L 2 1 ) was achieved over a 72-h period using 10 m M sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( < 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 6 26%. CHO-hLCATH6 and CHO- hLCAT species had identical specific activities (26 6 6 and 26 6 3 nmol CE formed m g 2 1 h 2 1 , respectively). The enzy- matic activity of CHO-hLCATH6 was stable at 4 8 C in excess of 60 days. Substrate saturation studies, using rHDL com- posed of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indi- cated that the app K m for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at < 2 m M substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT. —Chisholm, J. W., A. K. Gebre, and J. S. Parks. Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase. J. Lipid Res. 1999. 40: 1512-1519.

Human lecithin:cholesterol acyltransferase (LCAT) circulates in plasma bound to high density lipoproteins (HDL) and modulates the rate by which cholesteryl ester is transported to the liver. So far, little is known about the regulation of the expression of the LCAT gene. In this study we have defined the cis-elements, identified the trans-acting factors and demonstrated their functional effects and significance in determining transcriptional activity of the proximal LCAT promoter. Using deletion mutants having 5′ variable ends (from nucleotides -72 to -27), we have identified the presence of two non-consensus GC-rich regions that stimulate transcription in HepG2 and HeLa cells. These regions designated sites A (-29 to -47) and B (-49 to -65) contain the CCTCC core sequence which in electromobility shift analysis is critical for the formation of two DNA–protein complexes designated I and II. Site-directed mutagenesis suggests that both sites are equally important in promoter activity, and that cooperative interactions between both sites are not required for activity. Electromobility shift and supershift experiments using oligonucleotides spanning sites A and B identified Sp1 and Sp3 as the transcription factors interacting at these sites. To determine the significance and functional effects that Sp1 and Sp3 have in regulating LCAT promoter activity, we performed transfection experiments in Drosophila SL-2 cells as they lack endogenous Sp1 and Sp3. Sp1 but not Sp3 activates the human LCAT promoter and when Sp1 is co-transfected along with Sp3, Sp3 functions as a dose-dependent repressor of Sp1-mediated activation. These findings indicate that Sp1 is capable of trans-activating a reporter gene linked to the LCAT promoter containing Sp binding sites and suggests that the levels of Sp3 or the nuclear Sp1/Sp3 ratio may play an important role in determining the transcriptional activity of the LCAT promoter in vivo.—Hoppe, K. L., and O. L. Francone. Binding and functional effects of transcription factors Sp1 and Sp3 on the proximal human lecithin: cholesterol acyltransferase promoter. J. Lipid Res. 1998. 39: 969–-977.

Although White Carneau (WC) pigeons are known to be more susceptible to atherosclerosis than Show Racer (SR) pigeons, the reasons for this difference are not fully understood. While no major differences are known in the lipoprotein composition, a difference in the cholesteryl ester (CE) composition was reported. However, there is little information on the activity or specificity of lecithin:cholesterol acyltransferase (LCAT), the major source of plasma CE. In order to determine whether the esterification of cholesterol or other functions of LCAT are compromised in WC pigeons, we studied the various reactions catalyzed by LCAT in the two groups. The cholesterol esterification was found to be significantly lower in WC pigeons, whether assayed with exogenous or endogenous substrates. Furthermore, lyso phosphatidylcholine (PC) esterification and oxidized PC hydrolysis, two other reactions carried out by LCAT, were also lower in WC. We found evidence for the presence of an active lysophospholipase in pigeon plasma, and this activity was also lower in WC compared to SR. A significant increase in the FC/PC ratio, another reported atherogenic risk factor, was found in WC plasma. Because of the absence of other hydrolytic enzymes in pigeon plasma, LCAT may play an important role in the metabolism of oxidized PC generated during lipoprotein oxidation, and therefore a decrease in its activity in White Carneau pigeons may contribute to increased risk of atherosclerosis.—Liu, M., R. W. St. Clair, and P. V. Subbaiah. Impaired function of lecithin:cholesterol acyltransferase in atherosclerosis-susceptible White Carneau pigeons: possible effects on metabolism of oxidized phospholipids.

A method is described for the determination of the initial rate of cholesterol esterification in human plasma, based on the enzymatic determination of free cholesterol in the plasma before and after incubation at 37 degrees C. The cholesterol esterification rate was linear up to 40 minutes. In 18 normal male and 10 normal female subjects the cholesterol esterification rate was 91 +/- 15 (mean +/- SD) and 62 +/- 12 nmoles/hr/ml of plasma, respectively.