©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Esterification of Oxysterols by Human Plasma Lecithin-Cholesterol Acyltransferase (*)

Stefan E. Szedlacsek (§) , Erwin Wasowicz (¶) , Stefan A. Hulea , Hiro I. Nishida , Fred A. Kummerow , Toshiro Nishida (**)

From the (1) The Burnsides Research Laboratory, Department of Food Science, University of Illinois, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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-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-oleoyl monoesters. Unlike 27-hydroxycholesterol, 25-hydroxycholesterol, 7-hydroxycholesterol, 7-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.


INTRODUCTION

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) (LCAT)() catalyzes the transfer of the phosphatidylcholine acyl group at sn-2 position to cholesterol, forming lysophosphatidylcholine and cholesteryl ester (1, 2, 3, 4) . The cholesterol present in discoidal high density lipoprotein particles containing apolipoprotein (apo) A-I is preferentially esterified by the LCAT reaction (5) . Many studies have dealt with the specificity of LCAT for acyl donor phospholipids (2, 6, 7, 8, 9) . There are also studies relating LCAT specificity to the acyl acceptor. It has been shown that not only cholesterol but also various other sterols (10) , saturated long-chain alcohols (11) , and water (11, 12) can serve as the acyl acceptors. Although the involvement of LCAT in the esterification of 7-hydroxycholesterol (7-OH-C) was speculated (13) , no study has been carried out concerning LCAT-catalyzed esterification of oxysterols.

In the human body, oxysterols are derived from the diet primarily as products of cholesterol autoxidation (14) or produced endogenously (15). Some oxysterols are normal intermediates in the metabolic pathways; for instance, 7-OH-C is produced from cholesterol by liver microsomal 7-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis (16) . The interest in oxysterols is mainly due to their various biological effects: cytotoxicity, inhibition of -hydroxy--methylglutaryl CoA reductase activity, activation of acyl-CoA acyltransferase, and inhibition of low density lipoprotein binding, which have been extensively reviewed (17) . In addition, some oxysterols may be involved in the atherosclerotic processes (17) . Human aortic tissue is known to contain 27-hydroxycholesterol (27-OH-C)() as a free sterol (18, 19, 20, 21) and its monoesters (22) and diesters (23, 24) . The aortic contents of 27-OH-C were related to the severity of atherosclerosis (19, 21, 25) .

It has been speculated that the LCAT reaction promotes the reverse transfer of cholesterol (1) . The removal of oxysterols from peripheral tissues may also be promoted by the LCAT reaction. It is known that oxysterols in human plasma are found not only in the free form but also as fatty acyl esters (13, 24, 26, 27) . The esters of 27-OH-C were detected at more than five times higher concentrations than the esters of other dihydroxysterols (24) . In the present study, we examined the esterification, by the LCAT reaction, of oxysterols most frequently identified in human plasma. By using 27-OH-C, we investigated the factors influencing the formation of mono- and diesters.


EXPERIMENTAL PROCEDURES

Materials

Cholesterol (>99%), cholesteryl oleate (CE 18:1), cholesteryl linoleate (CE 18:2), cholesteryl palmitate (CE 16:0), cholesteryl pentadecanoate (CE 15:0), cholesteryl arachidonate (CE 20:4), cholestanetriol (Triol-C), 7-OH-C, cholesterol 5,6-epoxide (-epoxy-C), 20-hydroxycholesterol, apoA-I from human plasma (97%), dipalmitoyl-sn-glycero-3-phosphorylcholine (16:0/16:0-PC), dioleoyl-sn-glycero-3-phosphorylcholine (18:1/18:1-PC), dilinoleoyl-sn-glycero-3-phosphorylcholine (18:2/18:2-PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (18:0/20:4-PC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphorylcholine (16:0/18:2-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (16:0/18:1-PC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphorylcholine (18:1/16:0-PC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphorylcholine (18:0/18:1-PC), sodium azide, sodium cholate, and 2-mercaptoethanol were obtained from Sigma. Diarachidonoyl-sn-glycero-3-phosphorylcholine (20:4/20:4-PC) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (16:0/20:4-PC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 27-Hydroxycholesterol (27-OH-C), cholesterol 5,6-epoxide (-epoxy-C), and 7-OH-C were obtained from Research Plus, Inc. (Bayonne, NJ). Steraloids, Inc. (Wilton, NH) provided 7-ketocholesterol (7-keto-C), 25-hydroxycholesterol (25-OH-C) and 5-cholestene-25-hydroxy-3-oleate (25-OH-C[3]18:1). Fatty acid-free bovine serum albumin was purchased from Boehringer Mannheim. Bis(trimethylsilyl) trifluroacetamide and boron trifluoride in methanol (12%, w/w) were purchased from Supelco, Inc, (Bellefonte, PA). Ethyl alcohol, USP absolute, was obtained from Midwest Grain Co. (Pekin, IL). Acetonitrile, chloroform, hexane, and methanol of HPLC grade were purchased from Fisher Scientific (Fair Lawn, NJ).

Experiments were performed, unless otherwise stated, in 39 mM phosphate buffer (ionic strength 0.1, pH 7.4) containing 0.025% EDTA and 2 mM sodium azide.

Synthesis of 27-Hydroxycholesterol Palmitate Esters

The monoesters (25R)-R-cholestene-27-hydroxy-3-palmitate (27-OH-C[3]16:0) and 27-palmitate (27-OH-C[27]16:0) and the diester 3,27-dipalmitate (27-OH-C[3]16:0[27]16:0) were synthesized and purified essentially according to the method of Teng et al.(28) to use as standards for HPLC, GC, and mass spectrometry. The identity of the esters was confirmed by mass spectrometry.

Preparation of DBP

DBP preparations containing phosphatidylcholine and cholesterol or oxysterol were prepared essentially according to the method of Kato et al.(29) . An ethanol solution (17.5 µl) containing 0.1 µmol of cholesterol or oxysterol and 0.5 µmol of phosphatidylcholine was rapidly injected with a Hamilton microsyringe into 0.4 ml of phosphate buffer while stirring under a stream of N. After addition of 38 µl of 200 mM sodium cholate and 40 µl of apoA-I solution (2.5 mg/ml) (29) , the mixture (0.49 ml) was dialyzed at 4 °C under N against phosphate buffer with stirring for 2 days with four changes of 2 liters each of buffer. After dialysis, the solution was adjusted to 0.5 ml with phosphate buffer and used immediately for experiments. For some preparations, indicated by Dialysis-4 days (Fig. 3), the samples were kept in dialysis bags with stirring for 2 additional days.


Figure 3: Effect of dialysis period for DBP preparations on the LCAT reaction. DBP preparations containing 27-OH-C and dioleoyl-phosphatidylcholine were prepared according to the procedure given under ``Experimental Procedures.'' Dialysis periods of 2 and 4 days designate the samples used immediately after 2 days of dialysis and those kept in dialysis bags for 2 additional days, respectively, before the LCAT reaction. Incubation of the reaction mixtures was carried out as described in the legend to Fig. 1. Results are mean ±S.D. of four experiments. Filled bar, 27-OH-C; diagonally striped bar, 27-OH-C[3]18:1; vertically striped bar, 27-OH-C[3]18:1[27]18:1; openbar,total ester.



LCAT Reaction

LCAT was purified from human plasma according to the method described previously (29, 30) . The final purification of the enzyme was approximately 20,000-fold over the starting plasma. The LCAT reaction was carried out using 200 µl of DBP solution and purified LCAT with the amounts given in the text in the presence of 4 mM 2-mercaptoethanol and 5 mg of bovine serum albumin in phosphate buffer in the final volume of 500 µl as described previously (30) . The reaction was stopped by adding 2 ml of methanol to the incubation mixture and heating at 55 °C under nitrogen for 30 min. Deviation from this general procedure is given in the text.

Purification and Analysis of the Reaction Products

Extraction of Unreacted Oxysterols and of Oxysterol Esters

A known amount of the internal standard, cholesteryl pentadecanoate (CE 15:0) or cholesteryl palmitate (CE 16:0) in CHCl, was added to the incubation mixtures treated with methanol after the LCAT reaction. Lipids were extracted with 6 ml of hexane-chloroform (4:1, v/v) as described previously (30) .

Reversed Phase HPLC

HPLC analyses of lipid extracts obtained after the LCAT reaction were carried out in the reversed phase mode using a Waters Liquid Chromatography system with Adsorbshere C18 (5 µm) column, 150 4.6 mm (Alltech Associates, Inc.). For HPLC separation of the lipid extracts, mobile phase of acetonitrile:isopropyl alcohol (1:1, v/v) with a flow rate of 1.0 ml/min was used. For the analysis of free oxysterol and purification of monoesters, they were rechromatographed on the same HPLC column using a solvent mixture, acetonitrile:isopropyl alcohol (90:10, v/v), with a flow rate of 1.8 ml/min. The separated compounds were subjected to GC fatty acid analysis, GC, and mass spectrometry analyses.

Derivatization and Gas Chromatography

For quantitative analyses of cholesterol and oxycholesterol by GC, the samples were derivatized to form trimethylsilyl ethers in a similar manner to that described by Maerker and Unruh (31) . GC was performed on a Hewlett-Packard 5890 Series II GC equipped with a programmable cool-on column inlet for the capillary column, flame ionization detector, and a 30-m 0.25-mm inner diameter fused silica capillary DB5 column (J & W Scientific, Folsom, CA). For fatty acid analysis, oxysterol esters and cholesteryl esters were subjected to boron trifluoride-catalyzed methanolysis according to the method of Morrison and Smith (32) . Fatty acid methyl esters were analyzed with a Hewlett-Packard gas chromatograph (model 5790) equipped with a flame ionization detector and Supelcowax-10, 30-m 0.32-mm inner diameter fused silica column (Supelco, Inc., Bellefonte, PA).

Mass Spectroscopy

Mass spectra were recorded on an HP 5985B GC/mass spectrometry system operated in the electron impact mode (20 eV). Samples were introduced by the direct probe and distilled from the probe at 60-350 °C (30 °C/min).


RESULTS

Analysis of the Products of LCAT Reaction

The products of the esterification of cholesterol and 27-OH-C by the LCAT reaction with 1-palmitoyl-2-oleoyl-phosphatidylcholine were separated by HPLC (Fig. 1). DBP preparations containing the phosphatidylcholine and cholesterol or 27-OH-C were used as the substrate particles. The cholesterol esterification yielded cholesteryl oleate (panel A, peak 2) and cholesteryl palmitate (peak3). The esterification of 27-OH-C produced both monoesters and diesters. The monoesters, 27-OH-C[3]18:1 ((25R)-5-cholestene-27-hydroxy-3-oleate) (panelB, peak5) and 27-OH-C[3]16:0 (3-palmitate) (peak6), showed considerably lower retention time than the corresponding cholesteryl esters. The appearance of smaller peaks of palmitoyl esters of 27-OH-C and cholesterol as compared with corresponding oleoyl esters reflects considerably higher specificity of LCAT for the acyl group of phosphatidylcholines at sn-2 position than that at sn-1 position (2, 33, 34) . Quantitative data with respect to the positional specificities are presented in a later section (). The diesters of 27-OH-C produced were 27-OH-C[3]18:1[27]18:1 ((25R)-5-cholestene-3,27-dioleate) (panelB, peak7), 27-OH-C[3]16:0[27]16:0 (3,27-dipalmitate) (panelB, peak9), and the mixtures of 27-OH-C[3]18:1[27]16:0 (3-oleate, 27-palmitate) and 27-OH-C[3]16:0[27]18:1 (3-palmitate, 27-oleate) (panelB, peak8). These diesters gave considerably longer retention time than the monoesters. The retention times of the monoesters and the diesters that were obtained with a mobile phase of acetonitrile:isopropyl alcohol (1:1, v/v) are included in . In view of relatively close retention times of the monoesters, the second mobile phase of acetonitrile:isopropyl alcohol (9:1, v/v), was used to obtain better separation of monoesters (). The monoesters in this solvent system were collected for GC and mass spectrometry analyses. The diesters were collected in the first solvent system for the analyses.


Figure 1: HPLC separation of lipid extracts obtained from the reaction mixtures for esterification of cholesterol (A) and 27-OH-C (B) with 1-palmitoyl-2-oleoyl-phosphatidylcholine by LCAT. DBP preparations containing 40 nmol of cholesterol or 27-OH-C and 200 nmol of 1-palmitoyl-2-oleoyl-phosphatidylcholine were incubated for 1 h with 2.6 µg of LCAT in the final volume of 0.5 ml as described under ``Experimental Procedures.'' The lipid extract was applied to a column of Adsorbsphere C18 (150 4.6 mm). Isopropyl alcohol:acetonitrile (1:1, v/v) was used as the mobile phase, at the flow rate of 1.0 ml/min. The absorbance at 205 nm versus the retention time was plotted. The numbers1-9 label the peaks of cholesterol (peak 1), CE18:1 (peak 2), CE16:0 (peak 3), 27-OH-C (peak 4), 27-OH-C[3]18:1 (peak 5), 27-OH-C[3]16:0 (peak 6), 27-OH-C[3]18:1[27]18:1 (peak 7), 27-OH-C[3]18:1[27]16:0 with 27-OH-C[3]16:0[27]18:1 (peak8), 27-OH-C[3]16:0[27]16:0 (peak9), and CE 15:0 (internal standard) (IS).



The fatty acid composition of the mono- and diesters produced from 27-OH-C were verified by GC analysis of the fatty acid methyl esters. Oxysterol components of the esters were analyzed by GC analysis after saponification and silylation. The mass spectra data for mono- and diesters of 27-OH-C are included in Tables II and III, respectively. All fragment ions listed could be ascribed to relatively simple cleavage processes and were assigned on the basis of their analogy with published spectral data. The mass spectra analysis of monoesters showed that the hydroxyl group of 27-OH-C are esterified at the position of C-3 and not at the position of C-27. This conclusion is based on the following observations. The 3-monoesters exhibited a high abundance of [M-RCOOH] (). The synthetic 27-monoesters of palmitic acid exhibited a high abundance of [M-Sc-HO] and of [M-HO] (, Footnote c). Moreover, the 27-monoester exhibited [M-85] and [M-111] representing [M-HO-CH] and [M-HO-CH], respectively. These two ions are known to be produced from -ene-3-alcohols but not from their 3-esters (28, 35) . These two ions were absent in the spectra of the 3-monoesters of 27-OH-C obtained by the LCAT reaction ().

The mass spectra of palmitoyl diester of 27-OH-C obtained by the LCAT reaction (I) were identical with those of the diester obtained synthetically. The palmitoyl diester exhibited the highest abundance of [M-2RCOOH] while the oleoyl and linoleoyl diesters gave the highest abundance of [M-RCOOH-Sc]. Apparently the side chain became more susceptible for cleavage with unsaturated diesters. In the case of diesters of 27-OH-C with two different acyl groups (Fig. 1B, peak8), mass spectra data proved the presence of palmitoyl ([M-RCOOH], m/z 648, 19%) and oleoyl ([M-RCOO], m/z 623, 42%) groups.

LCAT Esterification of Different Oxysterols Incorporated into DBP

The esterification of various oxysterols by the LCAT reaction was investigated by using DBP containing dioleoyl-phosphatidylcholine and individual oxysterols. Dioleoyl-phosphatidylcholine was used instead of 1-palmitoyl-2-oleoyl-phosphatidylcholine to simplify the analysis of the esterification products. The HPLC analysis of lipid extracts from all incubation mixtures revealed that only 27-OH-C gave, in addition to one monoester peak, one diester peak. The retention times of the monoester and diester were 7.9 and 33.6 min, respectively, when analyzed with the mobile phase of acetonitrile:isopropyl alcohol (1:1, v/v) (). All other oxysterols gave the single peaks of monoesters with retention times as given in . No hydroxyl groups, other than 3 hydroxyl groups, of 25-OH-C, 7-OH-C, 7-OH-C, and Triol-C were esterified by the LCAT reaction. Only monoester formation can be expected from 7-keto-C, -epoxy-C, and -expoxy-C. The retention times of oleoyl monoesters of oxysterols () generally agree with the polarity of the sterol moieties.

Mass spectrometry analysis of oleoyl monoesters separated by HPLC revealed that all oxysterols tested produced 3-monoesters. The lack of molecular ions in electron impact spectra, because of facile elimination of fatty acids from -ene-3-esters, is well documented (36, 37) . The oleoyl monoesters of 27-OH-C, 25-OH-C, 7-OH-C, and 7-OH-C exhibited the highest abundance of [M-RCOOH] (). Furthermore, synthetically prepared 3-oleoyl monoester of 25-hydroxycholesterol (25-OH-C[3]18:1) gave identical fragmentation as obtained for the oleoyl monoester of 25-OH-C produced by the LCAT reaction. The monoester of 7-keto-C gave the highest abundance of [M-RCOO], and [M-RCOOH] was absent. This ion formation may be characteristic of 7-keto-C oleoyl monoester and indicates the 3-monoester formation. Oleoyl monoesters of both -epoxy-C and -epoxy-C gave the highest abundance of [M-RCOO-HO], which may be the characteristic ion produced from the 3 oleoyl monoesters of epoxy-C having an epoxy group between carbons 5 and 6. The oleoyl monoester of Triol-C gave the highest abundance of [M-RCOO-HO] and also a very high abundance of [M-RCOO-2HO], which indicated that the two hydroxyl groups at carbon 5 and 6 are easily eliminated as the oleoyl group esterified at 3-position.

The LCAT reaction with various oxysterols and dioleoyl-phosphatidylcholine was carried out primarily to characterize the esterification products and to determine the possibility of diester formation. Therefore, the incubation was performed at 37 °C for 1 h in the presence of excess enzyme to obtain as much product as possible. Even under such incubation conditions, the extent of esterification of oxysterol varied widely, ranging from 30 to 90%. When cholesterol was substituted for oxysterols, approximately 86% of cholesterol was esterified under identical conditions. Among oxysterols tested, 7-OH-C, 7-keto-C, and -epoxy-C gave the highest degree of esterification. 25-OH-C and 7-OH-C gave about 70% of esterification, and -epoxy-C yielded about 50% of esterification. Triol-C was not an effective substrate for the esterification, possibly reflecting the pronounced hydrophilicity of the sterol. 27-OH-C was unique among oxysterols as described in later sections, by giving lower degrees of esterification and yet substantial degrees of diester formation.

Time Dependence of the Esterification of 27-OH-C with 1-Palmitoyl-2-oleoyl-phosphatidylcholine by LCAT

The time course of the LCAT reaction for 27-OH-C is presented in Fig. 2, panelA, and that of the esterification of cholesterol is given in panelB for comparison. Almost half of the initial amount of 27-OH-C was esterified after 15 min as apparent from the decrease in free 27-OH-C and increase in total esters produced. The total amount of 27-OH-C esters formed reached a plateau at 1 h of reaction time. Although the initial rate of cholesterol esterification was greater than that of 27-OH-C, the overall esterification yields became similar for cholesterol and 27-OH-C after l h of reaction. For both 27-OH-C and cholesterol, the oleoyl esters were preferentially formed as compared with palmitoyl esters, reflecting the enzyme specificity (2, 33, 34) . The yield of palmitoyl esters was about one-tenth that of the oleoyl monoesters (Fig. 2, and ). Among the diesters produced, only the oleoyl diester of 27-OH-C was plotted. The amounts of this diester and other diesters produced are given in , and the formation of a very small amount of palmitoyl diester was already indicated (Fig. 1, peak9).


Figure 2: Time course of the LCAT reaction for 27-OH-C and cholesterol. DBP preparations containing 20 nmol of 27-OH-C (panelA) or cholesterol (panelB) and 100 nmol of 1-palmitoyl-2-oleoyl-phosphatidylcholine were incubated with 1.3 µg of LCAT in the final volume of 0.25 ml as described under ``Experimental Procedures.'' The enzymatic reaction was stopped by adding methanol to each incubation mixture at various time periods. Results are means of four separate experiments. Typical standard deviations for values of free and esterified forms greater than 10 nmol were less than 0.5 nmol and for values less than 10 nmol were a maximum of 1.2 nmol. PanelA shows 27-OH-C (⊡), 27-OH-C[3]18:1 (), 27-OH-C[3]16:0 (), 27-OH-C[3]18:1[27]18:1 (), and total esters (). PanelB shows cholesterol (⊡), CE 18:1 (), CE 16:0 (), and total esters ().



Factors Influencing the Esterification of 27-OH-C with Dioleoyl-phosphatidylcholine by LCAT

When DBP preparations containing 27-OH-C and dioleoyl-phosphatidylcholine were used as substrates for LCAT reaction, significant variations were noted in the amounts of monoesters and diesters produced depending upon the length of dialysis period (Fig. 3). When the DBP preparations dialyzed for 2 days were used immediately for the LCAT reaction, it gave about 43% of unreacted 27-OH-C, 55% of monoesters, and 2% of diesters. In contrast, the DBP kept in the dialysis bag for 2 additional days reduced the extent of esterification, giving about 70% of unreacted 27-OH-C. Furthermore, the diester formation was considerably greater than the monoester formation. It was observed that the increased dialysis of the DBP preparations results in the formation of the particles with considerably larger molecular weight. The DBP dialyzed for 2 days gave the apparent particle mass of 200 kDa upon gel permeation chromatography on Biogel A 1.5 m column. The 4-day dialysis gave the particle masses ranging from 800 kDa to 1.2 10 Da. The size of DBP preparations might have influenced the extent of esterification as well as the formation of mono- and diesters. When 27-OH-C and phosphatidylcholine containing both saturated and unsaturated acyl groups, such as 1-palmitoyl-2-oleoyl-phosphatidylcholine, were used to prepare DBP, no significant differences in the particle weight and in the esterification were observed by the increased dialysis period. Both preparations dialyzed for 2 and 4 days gave an apparent particle mass of approximately 200 kDa.

In order to determine other factors influencing the proportions of mono- and diesters in DBP preparations containing 27-OH-C and dioleoyl-phosphatidylcholine, we studied the effects of LCAT and DBP concentrations on the esterification (Fig. 4). An increase in the LCAT concentration from 0.5 to 2.5 µg/ml not only increased the esterification yield but also changed the relative amounts of mono- and diesters produced. At an LCAT concentration as low as 0.5 µg/ml, 7.5 nmol of 27-OH-C was esterified. A higher LCAT concentration, 5.38 µg/ml, gave an esterification of about 13 nmol of 27-OH-C. While the esterification at low concentrations yielded mainly monoesters, higher concentrations of LCAT produced preferentially diesters. The plateau of the monoester curve at LCAT concentrations higher than 2.5 µg/ml indicates that a small part of monoester (approximately 15% of total esters) cannot be converted into diesters. The effect of DBP concentrations on the esterification of 27-OH-C was studied at a LCAT concentration of 5.2 µg/ml, the highest concentration used in the experiment for Fig. 4 . Higher concentrations of DBP preparations resulted in lower yields of diester and higher yields of monoester, thus giving lower diester:monoester ratios (data not given). It was apparent that the LCAT:DBP ratio governed the yields of mono- and diesters.


Figure 4: Influence of LCAT concentration on the esterification of 27-OH-C in DBP. DBP preparations containing dioleoyl-phosphatidylcholine and 27-OH-C with a dialysis period of 4 days were used for the LCAT reaction. The reaction mixtures contained 40 nmol of 27-OH-C and 200 nmol of dioleoyl-phosphatidylcholine in 200 µl of DBP preparations and various amounts of LCAT in a final reaction volume of 0.5 ml. Reaction time was 1 h. Duplicate experiments and duplicate HPLC assays for each sample were carried out, and the mean values and the standard deviations refer to the four values thus obtained. , 27-OH-C[3]18:1; , 27-OH-C[3]18:1[27]18:1; , total esters.



LCAT Reaction with 27-OH-C and Various Phosphatidylcholines in DBP

This experiment was carried out to determine the effects of various phosphatidylcholines on the esterification of 27-OH-C, especially the formation of the diesters, by using the DBP preparations dialyzed for 2 days. The acyl donor activities of various phosphatidylcholines for the esterification of 27-OH-C were compared with the donor activities for the cholesterol esterification. All phosphatidylcholines tested were able to donate acyl groups to 27-OH-C (). With the majority of phosphatidylcholines tested, comparable degrees of esterification yields were obtained for 27-OH-C and cholesterol. Dipalmitoyl-phosphatidylcholine exhibited the lowest esterification of both 27-OH-C and cholesterol among various phosphatidylcholines tested. This reflects the limited fluidity of the bilayer structures containing saturated phosphatidylcholine (7) . The yield of esterification of 27-OH-C was high with 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1-stearoyl-2-oleoyl-phosphatidylcholine, and 1-palmitoyl-2-linoleoyl-phosphatidylcholine. With all phosphatidylcholine containing saturated acyl group at sn-1 position and unsaturated acyl group at sn-2 position, the acyl group at sn-2 was transferred preferentially. The distributions of these sn-1 and sn-2 acyl groups in the monoesters of 27-OH-C were similar to their distributions in cholesterol esters. The extent of the utilization of sn-1 acyl group by LCAT for cholesterol esterification was in agreement with previous results (2, 33, 34) . The diesters of 27-OH-C were also formed in variable proportions for each phosphatidylcholine tested. The highest level of diester formation occurred with dilinoleoyl-phosphatidylcholine. 1-Palmitoyl-2-oleoyl-phosphatidylcholine, 1-stearoyl-2-oleoyl-phosphatidylcholine, and 1-pal-mitoyl-2-linoleoyl-phosphatidylcholine gave considerably higher degrees of diester formation than dioleoyl-phosphatidylcholine under the conditions employed. Regardless of the types of phosphatidylcholine used for the esterification, only 3-monoesters were formed as indicated by the mass spectra data () already described.

DISCUSSION

In the present study, the esterification of a variety of oxysterols by LCAT was investigated. The LCAT reaction of oxysterols having more than one hydroxyl group, unlike that of cholesterol, requires the determination of the location and number of the hydroxyl groups esterified. The esterification of 3-hydroxyl group of all oxysterols suggests that this hydroxyl group satisfies the steric requirement for the transfer of an acyl group from the acyl-enzyme intermediate (3, 10) . The diester formation by the esterification of the side chain 27-OH group proceeded only after the esterification of the 3-hydroxyl group. Apparently, the 27-OH group that is located at the end opposite to the 3 ester oxygen can be positioned at the enzyme active site for the esterification after the monoester formation. Such an optimal positioning might have not been obtained with other dihydroxysterols, such as 25-OH-C, 7-OH-C, and 7-OH-C, for diester formation with dioleoyl-phosphatidylcholine.

The ratio between 3-monoesters and diesters produced from 27-OH-C as well as the yield of the esters seems to be influenced by the physical characteristics of the DBP such as the particle size. It is possible that the aggregation of DBP containing 27-OH-C and dioleoyl-phosphatidylcholine may reduce the surface flexibility of the substrate particles and interfere with the removal of products from the active site of the bound enzyme. This would increase the retention time of monoesters at the active site and would enhance the formation of diesters especially when enzyme concentration is high (Fig. 4). The highest LCAT concentration (5.4 µg/ml) used in this study is roughly the physiological concentration of LCAT in human plasma.

Significant amounts of diesters were formed with the DBP preparations containing 27-OH-C and phosphatidylcholine having saturated and unsaturated acyl groups at sn-1 and sn-2 positions, respectively, such as 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1-stearoyl-2-oleoyl-phosphatidylcholine, and 1-palmitoyl-2-linoleoyl-phosphatidylcholine (). We noted that as much as 25% of diesters were produced with DBP containing 27-OH-C and 1-palmitoyl-2-oleoyl-phosphatidylcholine upon increasing the enzyme concentration. It may be generalized that the 3-monoester of 27-OH-C produced at the active site of LCAT has two possibilities: 1) to leave the active site and return to the surface layer of DBP or 2) to position itself into the hydrophobic cavity of the enzyme, making the 27-hydroxyl group available for the diester formation. The pathway proposed in Fig. 5could explain the formation of both monoesters and diesters. The adsorption of the enzyme to lipid-water interface leads to accommodation or binding of phosphatidylcholine and 27-OH-C to the enzyme active site (step 1) and to the formation of the acyl enzyme, E-Ac (step 2). The acyl group is transferred to the bound 27-OH-C, thus producing the bound monoester (step 3). The enzyme-3-monoester complex could release the monoester and the free enzyme (step 4) or could lead to the formation of enzyme-3-monoester-phosphatidylcholine complex (step 5), which can be converted to an acyl-enzyme-3-monoester complex (step 6). The acylation of the bound 3-monoester by the acyl group of the enzyme complex could lead to the formation of the enzyme-bound diester (step 8), which is subsequently released from the enzyme (step 9). The diester formation (step 8) may require the flip-flop of the 3-monoester for the correct positioning of the 27-hydroxyl group near the active site of the acyl-enzyme. Alternatively, 3-monoester can be released from the acyl-enzyme transition complex upon displacement by 27-OH-C (step 7). Thus, 3-monoester formation could proceed through the cyclic reaction pathway (steps 3, 5, 6, and 7), by incorporating phosphatidylcholine (step 5) and 27-OH-C (step 7) and by releasing lysophosphatidylcholine (step 6) and 3-monoester (step 7).


Figure 5: Proposed mechanism for the formation of mono- and diesters of 27-OH-C by the LCAT reaction. E, enzyme; E-Ac, acyl-enzyme.



The LCAT reaction usually occurs at the lipid-water interface, and efficient transfer of the enzyme between substrate particles is required (38) . The transfer of the enzyme is preceded by the enzyme desorption, which could occur, for example, in step 4 (Fig. 5), leaving the product and also substrates at the lipid-water interface (38) . The cyclic reaction, therefore, would occur only during the periods that the enzyme is associated with substrate particles. The LCAT reaction may best be explained by an interrupted cyclic or chain reaction mechanism. The number of cycles occurring during the successful encounter between the enzyme and substrate particle may be determined by various factors such as the rate of removal of lysophosphatidylcholine from the interface and apolipoprotein distribution at the interface (38) . The chain reaction pathways are supposed to be energetically more advantageous as compared with the Michaelis-Menten type mechanism and are the sole contributor to the catalytic process occurring with alcohol dehydrogenase and other enzymes (39) . It was previously observed that LCAT exerts phospholipase A activity (2, 10, 11) . This activity, however, is relatively low and was thought to be due to the limited penetration of water to the active site of the enzyme for the hydrolysis of acyl-enzyme (3, 10) . The low activity could also be the result of the absence of the cyclic or chain reaction, which may require the presence of an efficient acyl acceptor such as cholesterol.

In the present study we have shown that all oxysterols detected in human plasma are esterified by LCAT to form 3-acyl esters. Among various oxysterols tested, 27-OH-C formed diesters by the LCAT reaction. This represents the first demonstration of the enzymatic formation of sterol diesters. It was previously reported that approximately 70% of 27-OH-C is present in the esterified form in human plasma (25) although the proportions of mono- and diesters are not known. We are currently investigating the extent of the formation of mono- and diesters of 27-OH-C under conditions similar to those of human plasma. The presence of 50-84% of 7-OH-C in the esterified form in human plasma (13) may reflect the presence of the 3-monoesters since no diester formation occurred with this sterol. Approximately 50% of 7-ketocholesterol was reported to exist in esterified form (27) . It is possible that major portions of the plasma oxysterol esters, like cholesteryl esters, are derived by the LCAT reaction. There exists the possibility that oxysterols may exert more pronounced atherogenic effects than cholesterol (17) . Then the LCAT reaction may represent an important step in the reverse transport of oxysterols, alleviating their toxic effects. The clarification of the processes of the reverse transport awaits future investigations.

  
Table: 0p4in Mixture of diesters, having two different acyl groups at different positions, with identical retention time.(119)

  
Table: 0p4in 367 (92.7), [M-RCOO-2HO].(119)

  
Table: 0p4in [M-RCOO].(119)

  
Table: The products of the LCAT reaction obtained from DBP preparations containing 27-OH-C or cholesterol and various phosphatidylcholines

DBPs containing 27-OH-C or cholesterol were incubated with LCAT as described under ``Experimental Procedures.'' The concentration of LCAT in the reaction mixture was 5.2 µg/ml, and the incubation time was 1 h. The distribution of the esters produced is given in mole percent with respect to the initial amount, 40 nmol, of 27-OH-C or cholesterol used for the LCAT reaction. Diesters of 27-OH-C obtained in detectable amounts are listed. DBP containing 16:0/16:0-PC was prepared at 37 °C and dialyzed at room temperature. Duplicate experiments were carried out. For each experiment, two HPLC assays were done, and four values obtained were used to obtain the mean values and the standard deviations.



FOOTNOTES

*
This work was supported by a grant from the Wallace Genetic Foundation (to F. A. K.), Grant HL-17597 from the National Institutes of Health, United States Public Health Service (to T. N.), and funds from the Illinois Agriculture Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Inst. of Biochemistry, Dept. of Enzymology, Bucharest 77748, Romania.

Present address: Agricultural University, Inst. of Food Technology, Wojska, Poznan 60624, Poland.

**
To whom correspondence should be addressed: Burnsides Research Laboratory, University of Illinois, 1208 W. Pennsylvania Ave., Urbana, IL 61801. Tel.: 217-333-1876; Fax: 217-333-7370.

The abbreviations used are: LCAT, lecithin-cholesterol acyltransferase; apoA-I, apolipoprotein A-I; CE, cholesteryl ester; 27-OH-C, 27-hydroxycholesterol ((25R)-5-cholestene-3,27-diol); 25-OH-C, 25-hydroxycholesterol (5-cholestene-3,25-diol); 7-OH-C, 7-hydroxycholesterol (5-cholestene-3,7-diol); 7-OH-C, 7-hydroxycholesterol (5-cholestene-3,7-diol); 7-keto-C, 7-ketocholesterol (5-cholestene-3-ol-7-one); -epoxy-C, cholesterol 5,6-epoxide (cholestan-5,6-epoxy-3-ol); -epoxy-C, cholesterol 5,6-epoxide (cholestan-5,6-epoxy-3-ol); Triol-C, cholestanetriol (cholestan-3,5,6-triol); SE, side chain.

27-Hydroxycholesterol now replaces 26-hydroxycholesterol previously used in most articles, because of its stereochemistry is firmly established.


ACKNOWLEDGEMENTS

We express our appreciation to J. Jerrell for mass spectrum determination.


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