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At least 19 recordsLinked to original sources
PubMed1996

The distal pathway of lipoprotein-induced cholesterol esterification, but not sphingomyelinase-induced cholesterol esterification, is energy-dependent.

The stimulation of the intracellular cholesterol esterification pathway by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. The esterification pathway can also be stimulated by hydrolysis of cell-surface sphingomyelin by the enzyme sphingomyelinase (SMase). In both cases, intracellular cholesterol transport to the cholesterol esterifying enzyme, acyl-CoA:cholesterol O-acyltransferase (ACAT), is thought to be critical, although the mechanism of cholesterol transport is not known. In this report, we explore two fundamental properties of the cholesterol esterification pathway, namely its dependence on energy and the effect of other treatments that block membrane vesicle trafficking. After the atherogenic lipoprotein, beta-very low density lipoprotein (beta-VLDL), was internalized by macrophages and hydrolyzed in lysosomes, the cells were depleted of energy by treatment with sodium azide and 2-deoxyglucose or by permeabilization. Under these conditions, which allowed equal beta-VLDL-cholesteryl ester hydrolysis, cholesterol esterification was markedly decreased in the energy-depleted cells. This effect was not due to blockage of lysosomal cholesterol export. In the permeabilized cell system, energy repletion restored beta-VLDL-induced cholesterol esterification. Remarkably, stimulation of cholesterol esterification by SMase was not inhibited by energy depletion. Energy depletion also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification in Chinese hamster ovary cells. Similar experiments were carried out using N-ethylmaleimide, low potassium medium, or inhibitors of phosphatidylinositol 3-kinase, each of which blocks intracellular membrane vesicle trafficking. These treatments also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification. Finally, we show here that SMase treatment of cells leads to an increase in plasma membrane vesiculation that is relatively resistant to energy depletion. In summary, the stimulation of cholesterol esterification by lipoproteins, but not by SMase, is energy-dependent, N-ethylmaleimide-sensitive, and blocked by both low potassium and phosphatidylinositol 3-kinase inhibitors. The affected step or steps are distal to cholesterol export from lysosomes and not due to direct inhibition of the ACAT enzyme. Thus, the mechanisms involved in lipoprotein-induced versus SMase-induced cholesterol esterification are different, perhaps due to the involvement of energy-dependent vesicular cholesterol transport in the lipoprotein pathway and a novel, energy-independent vesicular transport mechanism in the SMase pathway.

Animals↗
PubMed1983

beta-sitosterol: esterification by intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.

Rabbits were fed either 10% coconut oil, 10% coconut oil and 1% beta-sitosterol, 10% coconut oil and 1% cholesterol, or 10% coconut oil and 1% beta-sitosterol plus 1% cholesterol for 4 weeks. Microsomal membranes from intestines of animals fed the 1% beta-sitosterol diet had 48% less cholesterol and were enriched twofold in beta-sitosterol compared to membranes from animals fed the coconut oil diet alone. Acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in jejunum and ileum was decreased significantly in animals fed the plant sterol alone. In membranes from animals fed 1% beta-sitosterol and 1% cholesterol, beta-sitosterol content increased 50% whereas cholesterol was modestly decreased compared to their controls fed only cholesterol. Intestinal ACAT was unchanged in the animals fed both sterols when compared to their controls. beta-Sitosterol esterification was determined by incubating intestinal microsomal membranes with either [(14)C]beta-sitosterol-albumin emulsion or [(14)C]beta-sitosterol:dipalmitoyl phosphatidylcholine (DPPC) liposomes to radiolabel the endogenous sterol pool. Oleoyl-CoA was then added. The CoA-dependent esterification rate of beta-sitosterol was very slow compared to that of cholesterol using both techniques. An increased amount of endogenous microsomal beta-sitosterol, which occurs in animals fed 1% beta-sitosterol, did not interfere with the stimulation of ACAT activity secondary to cholesterol enrichment of the membranes. Enriching microsomal membranes three- to five-fold with beta-sitosterol did not affect ACAT activity. Freshly isolated intestinal cells were incubated for 1 hour with [(3)H]oleic acid and beta-sitosterol:DPPC or 25-hydroxycholesterol:DPPC. Incorporation of oleic acid into cholesteryl esters did not change in the presence of beta-sitosterol but increased fourfold after the addition of 25-hydroxycholesterol. We conclude that the CoA-dependent esterification rate of cholesterol is at least 60 times greater than that of beta-sitosterol. Membrane beta-sitosterol does not interfere with nor compete with cholesterol esterification. Inadequate esterification of this plant sterol may play a role in the poor absorption of beta-sitosterol by the gut.-Field, F. J., and S. N. Mathur. beta-Sitosterol: esterification by intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.

Acyl Coenzyme A↗
PubMedDate not supplied

[Esterification of cholesterol oxidation products and their effect on the rate of cholesterol esterification in macrophages].

Effects of cholesterol (CH) autooxidation products on incorporation of 14C-oleate into cholesteryl esters (CE) and their possible esterification were studied in cultivated mice peritoneal macrophages (MPM). 25 Mg/ml of purified CH and 25 Mg/ml of autooxidized CH stimulated (4- and 17-fold, respectively) the cholesterol esterification in MPM. In presence of 4 Mg/ml 25-hydroxy CH or the mixture of 4 Mg/ml 7 alpha-, 7 beta-hydroxy CH and 7-keto CH incorporation of oleate into cellular CE was increased 20- and 4-fold, respectively. 4 Mg/ml concentration of cholestane-3 beta, 5 alpha, 6 beta-friol caused no effects. Mono- and diesters of 14C-hydroxy CH were found after 8 hrs incubation of the steroid with MPM. Incorporation of 14C-25 hydroxy CH into its esters occurred at the rate, which exceeded 6-7-fold the 14C-CH incorporation into CE. Esterification of 7 alpha-, 7 beta-hydroxyCH and 7-ketoCH was studied, 0.52% label of the total cell sterols radioactivity was detected in the fraction of nonpolar steroids. 14C-cholestane-3 beta,5 alpha,6 beta-triol was not esterified in MPM. The stimulating effects of 25-hydroxyCH on oleate incorporation into CE and its esterification in MPM were inhibited in presence of 10 Mg/ml progesterone, an inhibitor of acyl-CoA:cholesterol acyltransferase activity. Induction of CE formation in MPM by means of choltransferase activity. Induction of CE formation in MPM by means of cholesterol oxidation products may be responsible for development of foam cells, while esterification of polar steroids in cells appears to be of importance in decrease of their toxic and metabolic effects.

Animals↗
PubMed1983

Increased methyl esterification of membrane proteins in aged red-blood cells. Preferential esterification of ankyrin and band-4.1 cytoskeletal proteins.

The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins has been investigated in three different age-related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine, the precursor in vivo of the methyl donor S-adenosylmethionine, a fourfold increase in membrane-protein carboxyl methylation was observed in the oldest cells compared with the youngest ones. The identification of methylated species, based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, shows, in all cell fractions, a pattern similar to that reported for unfractionated erythrocytes. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell ageing. Particularly in band 4.1 the age-related increase in methyl esterification is accompanied by a significant reduction of the half-life of methyl esters. The activity of cytoplasmic protein methylase II does not change during cell ageing, while the isolated ghosts from erythrocytes of different age show an age-related increased ability to act as methyl-accepting substrates, when incubated in presence of purified protein methylase II and methyl-labelled S-adenosylmethionine, therefore the relevance of membrane structure in determining membrane protein methylation levels can be postulated. Finally the possible correlation of this posttranslational protein modification with erythrocyte ageing is discussed.

Adult↗
PubMed1987

Endogenous esterification of bilirubin by liver microsomes. Evidence for an intramicrosomal pool of UDP-glucose and lumenal orientation of bilirubin UDP-glycosyltransferase.

Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.

Animals↗
PubMed1989

Comparison in inhibitory effects of lipolysis products on cholesterol esterification.

The effects of lipolysis products (glycerol, free fatty acids and lysolecithin) on cholesterol esterification in LDL and HDL3 were studied. The effects of oleic acid, linoleic acid and EPA on cholesterol esterification in LDL and HDL3 were also compared. 14C-FC labeled lipoprotein, LCAT source (lipoprotein deficient plasma) and test substance were incubated at 37 degrees C, and cholesterol esterification rates were estimated. In LDL, glycerol and palmitic acid did not inhibit cholesterol esterification. The inhibition rates of cholesterol esterification by lysolecithin were same as those by linoleic acid in LDL and increased to 100% at 2.5 mM depending on the concentrations. The effects of oleic acid, linoleic acid, and EPA were compared. The inhibition rates of cholesterol esterification in LDL were highest by EPA, next by linoleic acid and lowest by oleic acid. In HDL3, glycerol did not inhibit cholesterol esterification. Palmitic acid inhibited cholesterol esterification in HDL3. The inhibition rates of cholesterol esterification by lysolecithin in HDL3 were always lower than those by linoleic acid. The inhibition rates by palmitic acid were almost same as those by lysolecithin in HDL3. The inhibition rates of cholesterol esterification by EPA were higher than those by linoleic acid in HDL3. The inhibition rates of cholesterol esterification by oleic acid were close to those by linoleic acid in HDL3. Polyunsaturated FA suppressed LCAT activities much stronger than saturated FA at physiological concentrations.

Apolipoproteins↗
PubMed1995

Effect and cellular site of action of cysteine protease inhibitors on the cholesterol esterification pathway in macrophages and Chinese hamster ovary cells.

Stimulation of intracellular cholesterol esterification, which is catalyzed by the enzyme acyl-CoA:cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. Since other aspects of intracellular cholesterol metabolism involve proteolytic reactions, we looked for evidence of intracellular proteolysis in the stimulation of the cholesterol esterification pathway. When macrophages and CHO cells were incubated with the cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), the ability of beta-very-low-density lipoprotein (beta-VLDL) and free cholesterol-rich liposomes to stimulate cholesterol esterification was inhibited by 60-90%. Epoxysuccinylleucylamido-3-methylbutane ethyl ester (EST), a cysteine protease inhibitor structurally different from ALLN, also inhibited beta-VLDL-induced cholesterol esterification in CHO cells. The inhibitory effect of the protease inhibitors could not be explained by decreased net expansion of cellular cholesterol pools, inhibition of lipoprotein cholesteryl ester hydrolysis, or blockage of cholesterol trafficking through the lysosomal pathway. Furthermore, stimulation of cholesterol esterification by 25-hydroxycholesterol and sphingomyelinase was not inhibited by ALLN, indicating that ALLN is not acting as a direct ACAT inhibitor in the cells, and suggesting that the ALLN effect is specific for methods of stimulating cholesterol esterification that expand cellular cholesterol pools. Previous studies have shown that inhibition of protein synthesis (e.g., by cycloheximide) stimulates cholesterol esterification in macrophages and CHO cells, suggesting the presence of a short-lived protein inhibitor of cholesterol esterification. Herein, we show that, when added after cycloheximide, ALLN does not inhibit cycloheximide-induced cholesterol esterification in either cell type. The data in this report are consistent with a novel model in which a proteolytic reaction mediates the stimulation of cholesterol esterification specifically by expanded cellular cholesterol pools. The apparent protease-dependent step is not dependent upon lysosomal trafficking of cholesterol and is proximal to the ACAT enzyme itself; it may function by cleaving an endogenous inhibitor of the interaction of expanded cellular cholesterol pools with ACAT.

Animals↗
PubMed1980

Effect of insulin on in vitro intestinal fatty acid esterification in the rat.

We have previously shown that glucose metabolism plays an important role in modifying intestinal fatty acid esterification. Because it is well known that glucose metabolism is under insulin regulation, we examined the effect of insulin on intestinal fatty acid esterification. Insulin pretreatment led to a marked decrease in in vitro intestinal fatty acid esterification, but this decrease was abolished by maintaining blood glucose above 80 mg/dl. Addition of insulin to the incubation medium failed to produce any effect on intestinal fatty acid esterification. The decreased fatty acid esterification on hypoglycemic rats was not associated with changes in fatty acid uptake or lipid esterifying enzyme activities. However, there was a significant increase in the production of volatile metabolites of fatty acid. We conclude that 1) insulin itself has no effect on intestinal fatty acid esterification, 2) the effects observed in this study are due to insulin-induced hypoglycemia, 3) hypoglycemia does not alter intestinal fatty acid uptake or intrinsic esterification activity, but leads to preferential oxidation rather than esterification of fatty acid by the small intestine, and 4) the critical blood glucose concentration needed to maintain normal esterification in the rat was approximately at 80 mg/dl.

Animals↗
PubMed1977

Stimulation of cholesterol esterification in rhesus monkey arterial smooth muscle cells.

The influence of homologous high density lipoprotein (HDL) and low density lipoprotein (LDL) and of whole hypercholesterolemic serum on the esterification of oleic acid and cholesterol was studied in rhesus monkey arterial smooth muscle cells. Whole hypercholesterolemic serum and isolated LDL stimulated cholesterol esterification as much as 10-fold using either cholesterol-1,2-3H or oleate-1-14C as substrate. At the same concentrations of cholesterol, HDL stimulated cholesterol esterification to a lesser extent, to a maximum of 3-fold. Associated with the stimulation of cholesterol esterification by LDL or whole hypercholesterolemic serum was a greater than 10-fold increase in the cholesteryl ester content of the arterial smooth muscle cells. Esterification to cholesterol reached a maximum after 8-12 hours of culture with either hypercholesterolemic serum or LDL. The stimulation of esterification was specific for esterification to cholesterol because there was little change in incorporation of fatty acid into triglycerides and phospholipids. These studies provide further evidence that a major consequence of the interaction of plasma LDL with the cellular elements of the arterial wall is a stimulation of cholesterol esterification. These studies, coupled with the observation that cholesteryl esters, more than any other single component, increase in the atherosclerotic artery, suggest an important role of a stimulation in cholesterol esterification in the pathogenesis of atherosclerosis.

Animals↗
PubMed1995

A comparison of the esterification of steroids by rat lecithin:cholesterol acyltransferase and acyl coenzyme A:cholesterol acyltransferase.

Although fatty acid esters of several steroids have been found in both blood and tissues, their biosynthetic origins are uncertain. For example, the fatty acid esters of delta 5-3 beta-hydroxysteroids pregnenolone and dehydroepiandrosterone (DHEA) are synthesized in tissues by an acyl coenzyme A:acyltransferase. These esters are not secreted, and the circulating esters are formed in blood by lecithin:cholesterol acyltransferase (LCAT). Fatty acid esters of corticosterone (B) and estradiol (E2) are also present in both blood and tissues, but unlike the delta 5-3 beta-hydroxysteroids, their structures are so different from cholesterol that it would not necessarily follow that they are esterified by the same enzyme. We have examined the esterification of the steroids DHEA, B, and E2 in blood and tissue, in comparison to the esterification of cholesterol, using as a model plasma and hepatic microsomes from the rat. All of the steroids were esterified in plasma, but at very different rates: cholesterol > DHEA >> E2 = B. The LCAT inhibitor, 5.5'-dithiobis-(2-nitrobenzoic acid), inhibited the esterification of all of the substrates. DHEA inhibited the esterification of cholesterol, albeit only at high concentration. The fatty acid compositions of the cholesterol and DHEA esters were analyzed, and they were found to be identical, with arachidonate the predominant ester, greater than 60%. In hepatic microsomes, the rate of esterification was different than plasma: cholesterol > E2 > or = DHEA >> B. Although B was esterified in both plasma and hepatic microsomes, the rate was exceedingly slow in both. The acyl coenzyme A:cholesterol acyltransferase inhibitor, N'-(2,4-difluorophenyl)-N-[[4-(2,2-dimethylpropyl)phenyl]- methyl]-N-heptylurea, blocked the esterification of cholesterol almost completely, but surprisingly, it had no effect on the esterification of the other steroids. The fatty acid esters of cholesterol, E2, and DHEA synthesized in the hepatic microsomes were analyzed. The composition of the cholesterol esters from the microsomes was very different than the esters of DHEA and E2. These results show that all of the steroids tested are esterified by LCAT, and consequently that blood LCAT is the probable source of the circulating steroidal esters. Most interesting are the studies of microsomal esterification. It has been presumed that similar to blood, the esterification of steroids in tissues is carried out by the same enzyme that esterifies cholesterol.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Role of multidrug resistance P-glycoproteins in cholesterol esterification.

Cholesterol esterification, catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT), plays a central role in cellular cholesterol homeostasis and in physiologic processes that lead to coronary heart disease. Although ACAT resides in the endoplasmic reticulum (ER), the cholesterol substrate for esterification originates in the plasma membrane and must be transported to the ER for esterification. Progesterone inhibits esterification, possibly by blocking the transport of cholesterol to the ER. Recent studies suggest that progesterone acts by inhibiting the activity of one or more of the multidrug-resistant (MDR) P-glycoproteins. In the current manuscript, we demonstrate that progesterone's ability to inhibit esterification is not mediated through the progesterone receptor. We evaluate a series of steroid hormones and find a strong correlation between a steroid hormone's hydrophobicity and its ability to inhibit both cholesterol esterification and MDR-catalyzed drug efflux. We also find that cholesterol esterification is inhibited by nonsteroidal MDR inhibitors, and that this inhibition specifically affects the esterification of cholesterol derived from the plasma membrane. MDR inhibitors also inhibit cholesterol esterification in a wide range of cultured human cell lines. These observations suggest that MDR activity normally functions in a general process of intracellular cholesterol transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1981

Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

The relationship between plasma lecithin:cholesterol acyltransferase mass and enzyme activity and between mass and plasma cholesterol esterification rate was determined in 25 adult volunteers without overt disease (14 normolipidemic and 11 hyperlipidemic). Furthermore, the relationship of lecithin:cholesterol acyltransferase mass and cholesterol esterification rate to lipids, apoproteins, age, and ideal body weight was assessed. Lecithin:cholesterol acyltransferase mass determined by radioimmunoassay was highly correlated with enzyme activity assayed using a heated plasma substrate (r = 0.636) and with the molar cholesterol esterification rate determined either by radioassay (r = 0.809) or by measurement of the decrease of unesterified cholesterol (r = 0.621). Lecithin:cholesterol acyltransferase mass was also positively correlated with total cholesterol (r = 0.608), unesterified cholesterol (r = 0.562), age (r = 0.544), and percent ideal body weight (r = 0.619), but was not significantly correlated with log triglyceride, high density lipoprotein cholesterol, or apolipoproteins A-I, A-II, or D. Plasma cholesterol esterification rate by both methods was highly positively correlated with total cholesterol, unesterified cholesterol, log triglyceride, and age, but was inversely correlated with high density lipoprotein cholesterol. Upon partial correlation analysis with lecithin:cholesterol acyltransferase mass kept constant the cholesterol esterification rate remained significantly positively related to total cholesterol, unesterified cholesterol, and log triglyceride and inversely related to high density lipoprotein cholesterol. Two subjects had normal lecithin:cholesterol acyltransferase but approximately half normal molar cholesterol esterification rate. Measurement of lecithin:cholesterol acyltransferase mass and activity along with plasma cholesterol esterification rate will permit differentiation of abnormalities of enzyme from qualitative or quantitative substrate or cofactor abnormalities. Also, the finding that the regression line between LCAT mass and the plasma esterification rate by direct determination of unesterified cholesterol passes through the origin suggests that all immunodetectable LCAT in plasma is active in normal subjects.-Albers, J. J., C-H. Chen, and J. L. Adolphson. Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

Adult↗
PubMed1985

Retinol esterification in cultured rat liver cells.

Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells.

Acyltransferases↗
PubMed1995

Esterification of oxysterols by human plasma lecithin-cholesterol acyltransferase.

In the present study, lecithin-cholesterol acyltransferase (LCAT) catalyzed esterification of oxysterols was investigated by using discoidal bilayer particles (DBP) containing various oxysterols, phosphatidylcholines, and apolipoprotein A-I. The esterified oxysterols were analyzed by high pressure liquid chromatography, gas chromatography, and mass spectrometry. LCAT esterified all oxysterols tested that are known to be present in human plasma. The esterification yields in almost all cases were relatively high, often as high as the yield of cholesterol esterification. When DBP preparations containing 27-hydroxycholesterol and various phosphatidylcholines were used for the LCAT reaction, both monoesters and diesters were produced. The mass spectrometry analysis showed that the monoester was produced by the esterification of the 3 beta-hydroxyl group and not the 27-hydroxyl group. The diesters were apparently produced by the esterification of the 27-hydroxyl group only after the esterification of the 3 beta-hydroxyl group. Phosphatidylcholine containing a saturated acyl group at sn-1 position and an unsaturated acyl group at sn-2 position gave generally high esterification yield. The esterification of various oxysterols was compared by using DBP containing dioleoyl-phosphatidylcholine and individual oxysterols. All oxysterols produced 3 beta-oleoyl monoesters. Unlike 27-hydroxycholesterol, 25-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, or cholestanetriol did not produce diesters. Various factors influencing the formation of the monoesters and diesters from 27-hydroxycholesterol were investigated. When dioleoyl-phosphatidylcholine was used as the acyl donor, prolonged dialysis of DBP preparations and increase in the ratio of the enzyme concentration to substrate particle concentration increased the diester formation. Significant amounts of diesters were also produced by using 1-palmitoyl-2-oleoyl-phosphatidylcholine and other phosphatidylcholines as the acyl donors. By analyzing the conditions of monoester and diester formation, a scheme for the LCAT reaction pathway was proposed.

Apolipoprotein A-I↗
PubMed1999

Regiospecific esterification of estrogens by lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT), the enzyme that esterifies cholesterol in blood, also esterifies other steroids at the 3beta-hydroxyl. These steroids, like cholesterol, are delta5-3beta-hydroxysteroids, such as pregnenolone and dehydroepiandrosterone. One unusual LCAT substrate is the estrogen, estradiol, which is esterified at the 17beta-hydroxyl. The esterification of estradiol by LCAT has been reported to produce a powerful antioxidant that protects low density lipoprotein (LDL) from oxidation. We investigated the substrate specificity of LCAT, comparing the esterification of four different steroids (estradiol, estriol, testosterone, and 5-androstene-3beta, 17beta-diol) by human LCAT in blood and by acyl-coenzyme A:acyltransferase in tissue (placenta and fat). Estradiol was esterified only at the D ring 17beta-hydroxyl group in both blood and tissue. In contrast, although testosterone has a D ring structure identical to that of estradiol, and it was esterified at the 17beta-hydroxyl by acyl-coenzyme A:acyltransferase in tissue, it was not esterified by LCAT. When 5-androstenediol was the substrate in the tissues, both the 3beta- and 17beta-esters were synthesized, but the major product was the 17beta-ester. Conversely, although 5-androstenediol was an excellent substrate for LCAT, only the 3beta-hydroxyl was esterified. No 17beta-ester was formed. The comparison of the esterification of estriol by acyl-coenzyme A:acyltransferase and LCAT was also surprising. In the tissues, estriol is esterified at both D ring hydroxyls, and both are esterified about equally. Although estriol is an extremely polar estrogen, it is esterified by LCAT, albeit at a very slow rate. Although again both D ring hydroxyls were esterified, the LCAT esterification site was mainly at the 17beta-hydroxyl. Esterification of estriol at the 17beta-hydroxyl in preference to the 16alpha-hydroxyl is especially striking, because the 17beta-hydroxyl group is sterically shielded by the C-18 methyl group, making esterification at this position energetically much more difficult. Furthermore, these studies demonstrate that esterification of the 17beta-hydroxyl group by LCAT is unique to estrogens. It suggests that this unusual regiospecific esterification of C-17 of the estrogens underlies a distinct stereochemical requirement for the powerful antioxidant action that has reported for the estradiol esters formed by LCAT.

Acyl Coenzyme A↗