Net esterification in vitro of plasma cholesterol in human primary hyperlipidemia

Net esterification of cholesterol was studied in vitro in the plasma of normal subjects and of patients with different types of hyperlipidemia. Net esterification of cholesterol was found to be significantly enhanced in types IIa, IV and V of human primary hyperlipidemia. In the plasma of all normal and hyperlipidemic subjects taken as a group, net esterification of cholesterol is significantly correlated with free cholesterol (p<0.001) and with phosphatidyl choline (p<0.001).     

Plasma cholesterol turnover and esterification in the pigeon

The plasma cholesterol turnover and serum cholesterol esterifying system was studied in White Carneau pigeons. Eight pigeons received a single injection of 1,2-³H-cholesterol intravenously and the decline in plasma cholesterol specific activity was measured at intervals from 1–64 days. Kinetic analysis of the plasma cholesterol die-away curves indicates that plasma cholesterol turnover in the pigeon conforms to a 2 pool model. The mass of pool A (cholesterol in blood and those tissues which are rapidly equilibrated with blood) in pigeons maintaining consistently high serum cholesterol levels (≈900 mg/100 ml) was 988 mg and the daily fractional turnover of pool A was 19.7% compared with 676 mg and 12.2% found in pigeons maintaining consistently low (≈400 mg/100 ml) serum cholesterol levels. Serum cholesterol esterifying activity, in vitro, showed a positive correlation (rxy=0.806) with serum cholesterol concentration in the pigeon. The pigeon differs, in this regard, from the chicken, rat and rabbit in which a negative correlation has been reported.     

On the rate of cholesterol esterification in cord blood serum

Cholesterol esterification was studied in adult and cord serum by measureing the initial rate of lecithin-cholesterol acyl transferase (LCAT) activity. Cord serum had about one-third as much free and esterified cholesterol and about one-half as much LCAT as adult serum. When the adult LCAT activities are plotted against the individual's serum free cholesterol levels a straight line relationship results (0.101±.005% cholesterol esterified per min). Cord serum LCAT activities (.135±.0407% cholesterol esterified per min) in the main fall above the adult line. Our results show that cord serum can esterify cholesterol at a rate equal to or higher than adult serum when the LCAT activity is related to the amount of serum free cholesterol present.     

Higher levels of plasma cholesterol sulfate in patients with liver cirrhosis and hypercholesterolemia

An analytical method for the determination of cholesterol sulfate (CS) in plasma using gas-liquid chromatography was developed. We measured plasma CS concentrations in patients with liver cirrhosis and hypercholesterolemia as examples of disorders that involve aberrations in cholesterol metabolism. Patients with liver cirrhosis had plasma CS concentrations that were significantly higher than those of control subjects (444.6±51.7vs. 253.0±24.6 μg/dL, mean ±SE). The levels of other lipids were lower in cirrhotics, although the differences were not significant. There was no correlation between the levels of CS and sulfated bile acids in cirrhotic patients. CS levels in plasma were also higher in subjects with hypercholesterolemia (413.7±44.5 μg/gL) however, the ratio of CS to total cholesterol (TC) clearly differed between cirrhotics and hypercholesterolemic subjects (1.44±0.11×10⁻³,vs. 3.31 ±0.63×10⁻³;P<0.05). Both in subjects with hypercholesterolemia and in healthy controls, the CS/TC ratio was similar and CS accounted for roughly 0.14% of the TC concentration.     

The esterification of cholesterol in the yolk sac membrane of the chick embryo

The uptake of lipid from the yolk by the yolk sac membrane of the chick embryo is accompanied by the rapid esterification of a large proportion of the yolk cholesterol. This could arise from enhanced acyl-CoA:cholesterol acyltransferase (ACAT) activity and/or inhibition of cholesteryl ester hydrolase (CEH) activity. The activity of ACAT was therefore measured in microsomes obtained from yolk sac membranes at various stages of development. A high level of activity (up to 929 pmol of cholesteryl oleate formed per min per mg protein) was found during the second half of this period. Supplementation with exogenous cholesterol stimulated ACAT activity in microsomes obtained from the tissue at the earlier, but not at the later, stages of development suggesting that the enzyme became saturated with microsomal cholesterol as development proceeded. Correlating with this, the concentration of cholesterol in the microsomes increased 4-fold between 9 and 20 d of development. The activity of CEH was very low in the microsomes and could not be detected in the cytosolic fraction. The activity of a protein, which has been shown to function as an inhibitor of CEH, was found to be present at all stages of development. The high activity of ACAT, together with the low activity of CEH and an active CEH inhibitor protein is a combination well suited to promote an essentially unidirectional conversion of cholesterol to cholesteryl ester. This process may be a major determinant of the rate of lipid transfer from the yolk to the embryo.     

Influence of dietary fatty acid composition on cholesterol synthesis and esterification in hamsters

To investigate the effects of dietary fat quality on synthesis and esterification of cholesterol, Syrian hamsters were fed diets containing corn, olive, coconut or menhaden oils (10% w/w) with added cholesterol (0.1% w/w). After 3 weeks, animals were sacrificed 90 min following IP injection of³H₂O. Synthesis of free cholesterol and movement of free cholesterol into ester pools were measured from³H-uptade rate in liver and duodenum. Plasma total cholesterol and triglycerides levels were highest in coconut oil-fed animals, whereas hepatic total cholesterol and ester levels were elevated in olive oil-fed animals, as compared with all other groups. No diet-related differences were seen in duodenal cholesterol or total fatty acid content. In duodenum, uptake of³H per g tissue into cholesterol was greater compared with liver; however, within each tissue,³H-uptake into cholesterol was similar across groups. Notably,³H-uptake into cholesterol ester in liver was highest in menhaden oil-fed animals. These data suggest that menhaden fish oil consumption results in enhanced movement of newly synthesized cholesterol into ester as compared with other fat types.     

Cholesterol synthesis and esterification in isolated enterocytes: Regulation by cholesterol and cholestyramine feeding

The purpose of the present study was to investigate the physiological control of the main regulatory enzymes of cholesterol metabolism in isolated enterocytes obtained from chick duodenum, jejunum and ileum. Cholesterol feeding resulted in an inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate 5-pyrophosphate decarboxylase, while cholestyramine feeding increased reductase activity in all the regions studied and decarboxylase activity only in duodenum. Cholesterol feeding markedly increased acyl-CoA:cholesterol acyltransferase, but the effects of cholestyramine were less clear. The effects on transferase activity cannot be due to differences in the availability of acyl-CoA as exogenous substrate as no significant differences were found in acyl-CoA hydrolase activity after any of the dietary treatments. The effects of cholesterol feeding were related to changes in the cholesterol content of epithelial cells, whereas in the case of cholestyramine this relationship was less apparent.     

Vitamin E reduces cholesterol esterification and uptake of acetylated low density lipoprotein in macrophages

The effects of vitamin E on cholesteryl ester (CE) metabolism in 1774 cells were examined. Pretreatment of 1774 cells with vitamin E at concentrations above 50 μM significantly decreased acetylated low density lipoprotein (LDL)-induced incorporation of [¹⁴C]oleate into CF in cells in a dose-dependent manner. This was partly due to vitamin E Also significantly inhibiting the uptake of [³H]CE-labeled acetylated LDL by 1774 cells. A trend existed toward suppression of acyl-CoA:cholesterol acyltransferase (ACAT) activity in the cell lysate at high vitamin E concentration, but there was no effect on hydrolysis of CE. These data indicate that vitamin E reduces the uptake of modified LDL and suppresses ACAT activity, resulting in less cholesterol esterification in macrophages; a novel mechanism underlying the antiatherogenic properties of vitamin E.     

Inhibition of cholesterol esterification in macrophages and vascular smooth muscle foam cells: Evaluation of E5324, an Acyl-CoA cholesterol acyltransferase inhibitor

Cholesteryl esters (CE) comprise the principal lipid class that accumulates within macrophages and smooth muscle cells of the atherosclerotic lesion. Acyl-CoA cholesterol acyl-transferase (ACAT) is the major enzyme responsible for esterification of intracellular cholesterol. We evaluated the ability of E5324 (n-butyl-N″-[-2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxyl]-6-methyl-phenyllurea), a novel, orally absorbable ACAT inhibitor, to inhibit esterification of fatty acids to cholesterol and CE accumulation in macrophages and in smooth muscle cells. E5324 significantly inhibited cholesterol esterification in rat aortic smooth muscle cells and in macrophages. In addition, E5324 reduced the cellular mass of CE, the significant measure of the efficacy of drugs designed to modulate cholesterol metabolism. E5324 treatment of macrophages exposed to acetylated low-density lipoprotein reduced CE mass by 97%, and treatment of lipid-loaded smooth muscle cells reduced CE mass by 29%. Although free cholesterol increased approximately twofold, this free cholesterol would presumably be accessible to the membrane for effluxin vivo (reverse cholesterol transport). These results demonstrate that E5324 can inhibit cholesterol esterification and CE mass in atherosclerotic foam cells, derived from either macrophages or arterial smooth muscle cells.     

A model for studying LCAT reaction: In vitro cholesterol esterification in pig ovarian follicular fluid

Phosphatidylcholine acyltransferase (lecithin:cholesterol acyltransferase or LCAT; EC 2.3.1.43) activity was found to be present in pig ovarian follicular fluid (POFF), in addition to pig serum (PS). The cholesterol esterification rate in both POFF and PS is linear with incubation time up to 2 hr. The mean absolute rate of POFF-cholesterol esterification was 8.1±0.4 nmoles per ml per hr approximately one-fourth of that in PS. However, the fractional rate (percent of labeled cholesterol esterified per hr) of POFF-cholesterol esterification was similar to that observed in PS. There was little variation of absolute rate of cholesterol esterification in the fluid obtained from different sizes of follicles. Fatty acid or triacylglycerol did not participate in the reaction of cholesterol esterification in POFF. No appreciable change in enzymatic activity was found from storing POFF at 4 C for periods of time up to 24 hr or at −70 C up to 2 months, but activity was lost thereafter. On the other hand, PS showed a much longer period of stability (5 days at 4 C and 9 months at −70 C). A discrepancy between the fatty acid composition of cholesteryl esters formed by the LCAT reaction and the fatty acid composition at the C-2 position of phosphatidylcholine led us to propose a two-step mechanism for the LCAT reaction. It is concluded that the LCAT of POFF, as well as that of plasma, is specific for individual fatty acids rather than for the fatty acid composition of phosphatidylcholine. The fatty acid concentration of lysophosphatidylcholine decreased during prolonged incubation times (6 to 21 hr) suggesting that the increased lysophosphatidylcholine formed as a product of the LCAT reaction may be reused as substrate for the LCAT reaction or for hydrolysis by lysophosphatidylcholine hydrolase. Presented at the AOCS Meeting, New York, May 1977.     

Diurnal variation of plasma methyl sterols and cholesterol in the rat: Relation to hepatic cholesterol synthesis

Free cholesterol of plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) of the rat was high and that of plasma very low density lipoproteins (VLDL) was low during the dark period of the diurnal cycle. Variations in the esterified plasma sterols were inconsistent. Free methyl sterols were high in all lipoproteins during the dark phase. Simultaneously, the incorporation of¹⁴C-acetate into nonsaponifiable sterols and the concentrations of free methyl sterols and cholesterol in the liver were elevated.     

Synthesis of cholesterol esters in the plasma and liver of sheep

A study was made with sheep on the formation in vitro of long chain fatty acid esters of cholesterol by the lecithin-cholesterol-acyltransferase system present in the plasma and the acyl CoA-cholesterol-acyltransferase system present in the liver. The rate of cholesterol esterification in the plasma was 0.024 μmoles/ml/hr. The relative pattern of fatty acids esterified during incubation of the plasma remained constant over the 8 hr period of incubation and was similar to the fatty acids in the plasma cholesteryl esters before incubation began and to the fatty acids in the 2-position of the plasma lecithin. The predominant cholesteryl esters synthesized contained monoenoic and dienoic fatty acids. Unlike the bovine, there was no apparent discrimination in favor of the 18∶2 containing species of plasma lecithin as donors of fatty acids. This difference could be accounted for by the similarity in the 18∶2 content of the phospholipids present in the high density (density >1.062 and < 1.21) and the low density (density > 1.006 and <1.063) lipoprotein fractions of the sheep plasma. The possibility of some discrimination against 20∶4 during cholesterol ester synthesis in the plasma of the sheep cannot be excluded. In the liver, the predominant cholesteryl esters synthesized contained saturated and monoenoic fatty acids; cholesteryl linoleate was synthesized to a very much less extent. There was considerable similarity between the composition of the unesterified fatty acid fraction of the liver before incubation began and the fatty acid composition of the cholesteryl esters synthesized during incubation. Addition of sonicated suspensions of free fatty acids altered markedly the fatty acid pattern of the cholesteryl esters synthesized by the liver slices. From the evidence presented it is concluded that the cholesteryl esters in sheep plasma are syntheized mainly by the plasma lecithin-cholesterol-acyltransferase system. The results are discussed in relation to cholesterol esterification systems demonstrated in the plasma and liver of monogastric animals.     

Vapor-liquid equilibria in systems with esterification reaction

The article presents a review of phase equilibrium data for four-component systems involving ester synthesis and hydrolysis reactions (acid-alcohol-ester-water) systems. A brief characteristic of the experimental and calculated data is given.     

Effect of dietary cholesterol protected against ruminal hydrogenation on the plasma cholesterol and liver of sheep

Dietary supplements containing cholesterol or sunflower oil were prepared to protect them against degradation in the rumen. On feeding daily supplements containing 1–2 g protected cholesterol and/or 100g protected sunflower oil to sheep, along with a basal ration of crushed oat grain and lucerne chaff, a rise in the plasma cholesterol was observed when compared with control animals. Livers from sheep fed protected cholesterol were enlarged, friable and cirrhotic in appearance and contained large deposits of esterified and free cholesterol, while livers from animals fed protected sunflower oil alone contained much less cholesterol. Octadecenoates constituted the major fatty acids in cholesterol esters, which, in animals fed protected sunflower oil, were mainly polyunsaturated. The factors involved in the deposition of liver lipid at very low dietary cholesterol concentrations (0.11–22%) in sheep compared with monogastric animals are discussed. Deceased.     

抗体芯片技术检测乳腺癌患者血清中PAI-1的表达

目的采用抗体芯片技术检测乳腺癌患者血清中纤溶酶原激活物抑制因子-1(plasminogen activator inhibitor,PAI-1)的表达。方法制备鼠抗人PAI-1单抗腹水及多抗,经ProteinA-Sepharose-4B柱纯化后,点至醛基玻片上(点样浓度均分别为1和0.1 mg/ml),制备抗体芯片,加入待测血清(分别进行1∶2、1∶10、1∶100稀释),利用晶芯Luxscan10K-A芯片扫描仪扫描,芯片图象分析软件晶芯SpotData Pro分析后,获得最适点样浓度和血清稀释倍数。采用优化后的抗体芯片法检测202份乳腺癌患者和204份健康体检者血清样本中PAI-1的表达。结果确定点样单抗最适浓度为1 mg/ml,血清最适稀释倍数为1∶100。PAI-1在204份健康体检者血清样本中阳性率为4.4%(9/204),在202份乳腺癌患者血清中的总体阳性率为25.2%,特异性为96%;其中119份发生转移和83份未发生转移的乳腺癌患者血清中PAI-1阳性率分别为35.3%(42/119)和10.8%(9/83),阳性率随肿瘤的分期进展而升高。结论 PAI-1在乳腺癌患者血清中的表达明显高于健康体检者,且随着肿瘤的分期进展阳性率升高,其有望成为新的乳腺癌血清筛查指标。     

云芝糖肽对乳腺癌患者外周血单个核细胞Toll样受体4的作用

目的探讨云芝糖肽(polysaccharopeptide,PSP)对乳腺癌患者外周血单个核细胞(peripheral blood mononuclearcell,PBMC)Toll样受体4(Toll-like receptor 4,TLR4)的作用。方法体外分离乳腺癌患者或健康人PBMC,并将其分别分为PSP组(分别加入终浓度为25μg/ml的PSP和终浓度为100μg/ml的PHA)和对照组(只加入终浓度为100μg/ml的PHA),采用TLR4单克隆抗体对各组细胞进行免疫荧光染色。将乳腺癌患者PBMC分为空白对照组、TLR4抗体组、PSP组和PSP+TLR4抗体组,采用Q-PCR检测各组PBMC IL-12、IL-6和TNF-α的表达水平。结果健康志愿者对照组PBMC荧光强度较强,而健康志愿者PSP组PBMC荧光强度较弱;乳腺癌患者PSP组与乳腺癌患者对照组相比,细胞荧光强度更弱。与空白对照组比较,TLR4抗体组PBMC IL-12和组TNF-α的表达水平均显著降低(P<0.05),PSP组PBMC IL-12、IL-6和TNF-α的表达水平均显著上调(P<0.05或<0.01);与PSP和TLR4抗体组比较,PSP+TLR4抗体组PBMC IL-12、IL-6和TNF-α的表达水平均显著上调(P<0.05或<0.01)。结论 TLR4可能是PSP的作用受体之一。     

The effect of a non-absorbable fat on the turnover of plasma cholesterol in the rat

Rats were injected with [4-¹⁴C]-cholesterol and then fed diets that contained sucrose polyester (SPE) at levels of 0 and 8% of the diet.¹⁴C was measured in neutral and acidic steroid fractions of the feces collected during days 35–39 post i.v. injection. Periodic blood samples were used to measure the specific activity of the plasma cholesterol. The plasma data were consistent with a two-pool model for the decay of the plasma specific activity. The slow component of the decay curve decreased more rapidly in animals that received SPE. The half-life corresponding to this component was approximately 20% shorter in the SPE-fed animals compared to the control group. The mass of cholesterol calculated for the first pool was similar for all groups of animals. The¹⁴C found in the feces was consistent with the more rapid removal of cholesterol from the body in the SPE-fed animals. The mass of excreted steroid was equal to the calculated rate of cholesterol production in each group of animals.     

Effect of dietary cholesterol oxidation products on the plasma clearance of chylomicrons in the rat

Oxidized cholesterols in the diet have been shown to exacerbate arterial cholesterol deposition and the development of atherosclerosis in animal models. Dietary oxidized cholesterols are absorbed through the intestine and incorporated into lymph chylomicrons. The aim of this study was to investigate the effect of oxidized cholesterols on the metabolism of nascent chylomicrons in vivo. It was shown that oxidized cholesterols markedly delay the clearance of chylomicrons from plasma compared to rats given TG alone. However, there was no difference in the clearance of chylomicrons containing oxidized cholesterols vs. purified cholesterol, although the presence of oxysterols did appear to exacerbate the removal of these particles from circulation. The impaired clearance of chylomicrons containing oxidized cholesterols was not due to impaired lipolysis and slower conversion to the remnant form. Moreover, the incorporation of oxidized cholesterols did not alter the hepatic or splenic uptake of chylomicrons compared to chylomicrons isolated from rats given purified cholesterol or TG alone. Collectively, the results of this study suggest that the exacerbated delay in clearance of chylomicron remnants enriched with oxysterols may be due to impaired uptake by tissues other than the liver and spleen. Apolipoprotein (apo) analysis showed that oxysterol incorporation reduced the apoE content and altered the apoC phenotype of chylomicrons, which may have an impact on the removal of chylomicron remnants from plasma. In conclusion, dietary oxysterols appear to have the potential to adversely affect chylomicron metabolism. Therefore, further investigations in humans are required to determine whether dietary oxidized cholesterols found in cholesterol-rich processed foods delay the clearance of postprandial remnants, which may contribute to and exacerbate the development of atherosclerosis.