Molecular Species Of Cholesteryl Esters Research Articles

Silibinin Suppresses the Hyperlipidemic Effects of the ALK-Tyrosine Kinase Inhibitor Lorlatinib in Hepatic Cells.

The third-generation anaplastic lymphoma tyrosine kinase inhibitor (ALK-TKI) lorlatinib has a unique side effect profile that includes hypercholesteremia and hypertriglyceridemia in >80% of lung cancer patients. Here, we tested the hypothesis that lorlatinib might directly promote the accumulation of cholesterol and/or triglycerides in human hepatic cells. We investigated the capacity of the hepatoprotectant silibinin to modify the lipid-modifying activity of lorlatinib. To predict clinically relevant drug–drug interactions if silibinin were used to clinically manage lorlatinib-induced hyperlipidemic effects in hepatic cells, we also explored the capacity of silibinin to interact with and block CYP3A4 activity using in silico computational descriptions and in vitro biochemical assays. A semi-targeted ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS/MS)-based lipidomic approach revealed that short-term treatment of hepatic cells with lorlatinib promotes the accumulation of numerous molecular species of cholesteryl esters and triglycerides. Silibinin treatment significantly protected the steady-state lipidome of hepatocytes against the hyperlipidemic actions of lorlatinib. Lipid staining confirmed the ability of lorlatinib to promote neutral lipid overload in hepatocytes upon long-term exposure, which was prevented by co-treatment with silibinin. Computational analyses and cell-free biochemical assays predicted a weak to moderate inhibitory activity of clinically relevant concentrations of silibinin against CYP3A4 when compared with recommended (rosuvastatin) and non-recommended (simvastatin) statins for lorlatinib-associated dyslipidemia. The elevated plasma cholesterol and triglyceride levels in lorlatinib-treated lung cancer patients might involve primary alterations in the hepatic accumulation of lipid intermediates. Silibinin could be clinically explored to reduce the undesirable hyperlipidemic activity of lorlatinib in lung cancer patients.

Multivariate Analysis for Molecular Species of Cholesteryl Ester in the Human Serum.

Cholesteryl ester (CE) is an ester of cholesterol and fatty acid (FA). Plasma CE reflects complicated metabolisms of cholesterol, phospholipids, lipoproteins, and dietary FAs. An informatics approach could be useful for analysis of the CE species. In this study, two basic dimension reduction methods, principal component analysis (PCA) and factor analysis, were applied to serum CE species determined by LC-MS/MS in a Japanese population (n = 545). PCA and factor analysis both reflected the size (concentration), food source, fat solubility, and biological aspect of the CE species. In a comparison between PCA (PC4) and factor analysis (factor 4), the latter was found to be more suggestive from a biological aspect of n-6 FAs. Cholesteryl docosahexaenoate (DHA) was found to be unique by a factor analysis, possibly relevant to the unique accumulation of DHA in the brain. An informatics approach, especially factor analysis, might be useful for the analysis of complicated metabolism of CE species in the serum.

Molecular species of cholesteryl esters formed via plasma lecithin: Cholesterol acyltransferase in fish oil supplemented dogs

Effects of a dietary supplement containing n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), on molecular species of cholesteryl ester (CE) formed via the plasma lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) reaction was studied. A commercially available basal diet was initially fed to 18 normal adult dogs for 10 days prior to supplementing the dogs, divided into two groups of nine, using either menhaden fish (MHO) or safflower (SAF) oil capsules for a 22 day period. Fatty acid compositions of plasma phospholipid (PL) and CE were measured at days 0, 10 and 32. Characterization of the plasma LCAT-derived reaction products formed in vitro was performed using a radiolabeled cholesterol substrate and molecular species formed were separated by argentation thin-layer chromatography. Significant differences in PL EPA, DHA, and docosapentaenoic acid (DPA) were found in the MHO group. The CE fatty acid distributions revealed a greater than 20-fold elevation in EPA and a nearly 6-fold increase in DHA after MHO supplements. However, DPA was not detected in any CE fatty acid samples suggesting that this fatty acid is not a substrate for cholesterol esterification. The CE molecular species in plasma and those formed via the plasma LCAT reaction in vitro support the possibility that LCAT is responsible, at least in part, for the plasma CE fatty acid distribution. Ratios of selected plasma CE and LCAT derived CE n-6 and n-3 fatty acids using the MHO group data resulted in 3-fold elevations of both DHA/EPA and DHA/AA in the LCAT-derived in vitro CE fraction compared to CE in whole plasma. It is concluded that while both EPA and DHA are suitable substrates for CE formation, relatively lower amounts of plasma CE-DHA compared to the LCAT derived CE-DHA indicates that this fatty acid may be selectively taken up by the tissues compared to CE-EPA and AA.

Oxidative degradation of cholesteryl esters in low-density lipoproteins: analysis by liquid chromatography-light scattering and protection by a new synthetic antioxidant, S20478.

Cholesteryl esters in the hydrophobic core of low-density lipoprotein (LDL) particles constitute a major molecular target during copper-mediated oxidation. To facilitate the rapid analysis and quantitation of the oxidative degradation of LDL cholesteryl esters, we describe a new approach based on light scattering detection following separation by HPLC. We have applied this approach to the evaluation of the protective capacity of a new synthetic antioxidant, S20478, during oxidation of LDL in the presence of copper ions. HPLC separation of cholesterol and the four major molecular species of cholesteryl esters (C16:0, C18:1, C18:2 and C20:4) of LDL was achieved in a single run of 20 min with high sensitivity (50 ng) and low background. Time course studies of the oxidative modification of LDL (ratio LDL protein: copper, 100 micrograms/mL: 1 microM) revealed that the content of unsaturated cholesteryl esters (C20:4 and C18:2) decreased (-30% and -15%, respectively) within 90 min of copper-mediated oxidation, while only minor degradation (up to 15%) of monounsaturated (C18:1) and saturated (C16:0) esters occurred. At 24 hours of oxidation, only traces (< 5%) of the C20:4 and C18:2 esters were detectable; whereas 52% of the C18:1 ester remained (P < 0.01). Of the saturated esters, only minor proportions (35% or less) underwent oxidative modification. In addition, some 81% of free cholesterol was conserved as the native sterol. The synthetic antioxidant, S20478 (50 microM) was capable of inhibiting the initiation and the propagation of copper-mediated LDL oxidation as determined by the time- and dose-dependent inhibition of the formation of conjugated dienes and thiobarbituric acid-reactive substances, as well as the conservation of the net electrical charge of LDL; indeed S20478 conserved cholesteryl esters in their native form up to 24 hours. However, after prolonged exposure to copper ions (48 hours), only 47% of the unsaturated esters remained (C18:2, P < 0.05). Nonetheless, S20478 (10 microM) was more efficient in inhibiting copper-mediated LDL oxidation as compared to probucol at the same concentration. These findings suggest that S20478 may be of potential interest in a new antioxidant approach to therapeutic stabilisation and regression of atherosclerotic plaques. Moreover, this method should prove useful in the assessment of the integrity of native LDL, and provides a new chemical marker of the degree of LDL oxidation.

Molecular species of cholesteryl esters formed in abetalipoproteinemia: effect of apoprotein B-containing lipoproteins.

In order to study the effects of very low density (VLDL) and low density (LDL) lipoproteins on the activity and specificity of lecithin:cholesterol acyltransferase (LCAT), we determined the molecular species of cholesteryl esters (CE) synthesized in the plasma from three abetalipoproteinemic (ABL) patients, before and after supplementation with normal VLDL or LDL. The patients' plasma had significantly lower concentration of 18:2 CE and higher concentrations of 16:0 CE and 18:1 CE compared to normal plasma. Incubation of ABL plasma with [4-14C]cholesterol at 37 degrees C and the subsequent analysis of labeled CE formed by high performance liquid chromatography revealed that the major species formed was 16:0 CE (34% of total label), whereas similar incubation of the d greater than 1.063 g/ml fraction of normal plasma resulted in the formation of predominantly 18:2 CE (45% of total label). Addition of normal VLDL or LDL to ABL plasma stimulated the total LCAT activity by 30-80% and normalized the CE species synthesized. The LCAT activity of a normal d greater than 1.063 g/ml fraction also was stimulated by the normal VLDL or LDL, but there was no alteration in the species of CE formed. Most of the CE synthesized was found in the added VLDL or LDL with both ABL and normal plasma, indicating that the CE transfer (CET) activity was not affected in ABL plasma. These results suggest that while the VLDL and LDL are required for the maximal activity of LCAT, the species of CE formed are primarily determined by the molecular species composition of phosphatidylcholine in the plasma.

Specificity of lipid transfer protein for molecular species of cholesteryl ester.

The capacity of the plasma-derived lipid transfer protein to facilitate the transfer of various cholesteryl ester species has been investigated. Four different molecular species of cholesteryl ester were incorporated into either reconstituted high density lipoproteins or phosphatidylcholine liposomes, and the resulting particles were used as donors in standardized lipid transfer assays. With reconstituted high density lipoproteins as substrate, the rate of transfer of cholesteryl esters was cholesteryl oleate greater than cholesteryl linoleate greater than cholesteryl arachidonate greater than cholesteryl palmitate. The transfer rate for cholesteryl oleate was 154% of that for cholesteryl palmitate. Liposome substrates gave similar results. It is concluded that lipid transfer protein transfers all major species of cholesteryl ester found in plasma; however, the relative rates of transfer were significantly affected by acyl chain composition. The transfer rates appeared to reflect substrate specificity rather than substrate availability within the donor particle.

Alterations in molecular species of cholesterol esters formed via plasma lecithin-cholesterol acyltransferase in human subjects consuming fish oil

The influence of a dietary supplement of n-3 polyunsaturated fatty acids containing eicosapentaenoic acid (EPA, 20: 5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on the molecular species of cholesteryl esters (CE) formed via the plasma lecithin (phosphatidylcholine) -cholesterol acyltransferase (LCAT; EC 2.3.1.43) reaction was evaluated. For this purpose, one group of eight subjects received an encapsulated fish lipid concentrate (MaxEPA) and another group of eight volunteers in the control group received encapsulated olive oil for 22 days. Plasma lipid profiles and fatty acid compositions of plasma phosphatidylcholine (PC) and CE were measured at day 0 and day 22 in all subjects. A decrease in plasma triglyceride (by 34%) and a moderate rise in high-density lipoprotein (HDL)-cholesterol (by 13%) was observed in the MaxEPA group. For characterization of the plasma LCAT-derived reaction products formed in vitro, [ 14 C]cholesterol was used as the substrate and the newly formed molecular species of [ 14C]CE were separated by argentation thin-layer chromatography. Marked shifts were found in the abundance of the various classes of LCAT-derived products in the MaxEPA group whereas no significant changes were observed in the controls. The proportion of the [ 14C]CE as pentaenoic (EPA) species rose by 9-fold (from 1.5% at day 0 to 14.4% at day 22) as the dienoic (linoleate) species fell (from 50.6 to 39.2%); a moderate rise in the hexaenoic (DHA) species (from 1.7 to 2.4%) with no significant change in the tetraenoic (arachidonate) (AA) species was observed. The LCAT results were in the order of the observed shifts in the fatty acid patterns of the plasma CE. The relative formation of different molecular species of [ 14C]CE via the plasma LCAT reaction as measured in vitro in the MaxEPA consumers was significantly different from the relative mol% distributions of the corresponding polyunsaturated fatty acids in plasma PC thereby suggesting the relative order of participation in the LCAT reaction in the MaxEPA group to be EPA > AA > DHA. The potential physiological significance of these findings is discussed in relation to EPA/DHA and HDL-cholesterol metabolism and function including cellular eicosanoid synthesis.