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Original Studies |
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06510
Address all correspondence and requests for reprints to: Dr. Richard B. Hochberg, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06510. E-mail: richard.hochberg{at}yale.edu
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Abstract |
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5-3ß-hydroxysteroids, such as pregnenolone and
dehydroepiandrosterone. One unusual LCAT substrate is the estrogen,
estradiol, which is esterified at the 17ß-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-3ß,17ß-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 17ß-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
17ß-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 3ß- and 17ß-esters were synthesized, but the
major product was the 17ß-ester. Conversely, although
5-androstenediol was an excellent substrate for LCAT, only the
3ß-hydroxyl was esterified. No 17ß-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
17ß-hydroxyl. Esterification of estriol at the 17ß-hydroxyl in
preference to the 16
-hydroxyl is especially striking, because the
17ß-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 17ß-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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-3ß-hydroxysteroids) as cholesterol
and are esterified at the 3ß-hydroxyl, for example,
dehydroepiandrosterone (DHEA), are esterified by a
different enzyme. This contrasts with lecithin:cholesterol
acyltransferase (LCAT), the enzyme in blood that catalyzes the
transesterification of the fatty acyl group from the 2'-position of
lecithin to the 3ß-hydroxyl of cholesterol, leading to the synthesis
of cholesterol esters. LCAT esterifies both cholesterol and a limited
number of other steroids. Although the acyl-CoA:acyltransferase(s) in
cells is capable of esterifying a wide array of steroids (3), there are
only a few known substrates for LCAT. They are
5-3ß-hydroxysteroids [pregnenolone,
DHEA, and 5-androstene-3ß,17ß-diol (Adiol)] and the
estrogen, estradiol (E2) (4). LCAT esterification of
steroids, especially of 5-3ß-hydroxysteroids, might
appear to be a fortuitous circumstance emanating from their structural
similarity to cholesterol. However, E2 is esterified at the
C-17 hydroxyl in the D ring (5, 6), the opposite end of the molecule
from the 3-hydroxyl (the esterification site of cholesterol). The
17ß-hydroxyl is not only far removed from the C-3 hydroxyl, but it is
also adjacent to and sterically shielded by the C-18 methyl group.
Consequently, the 17ß-hydroxyl is esterified only with difficulty,
illustrating that the LCAT esterification of E2 cannot be
rationalized simply by a structural similarity to cholesterol. Recently, it has been shown that the esterification of E2 in blood might serve an important physiological function in the prevention of heart disease (7). It is well known that men and postmenopausal women have a higher incidence of coronary artery disease compared to premenopausal women (8, 9). The increased susceptibility to heart disease of both men and menopausal women is thought to be due to a lack of estrogen. In fact, postmenopausal women receiving estrogen replacement therapy have a decreased risk of cardiovascular disease (10, 11). The antiatherogenic effect of estrogens is known to be complex, involving changes in the blood lipid profile as well as effects on blood vessels, etc. Another role estrogens may play in preventing heart disease is in the inhibition of low density lipoprotein (LDL) oxidation, a primary step in atheromatous plaque formation (12). Several studies have demonstrated that estrogens directly inhibit the oxidation of LDL (13, 14, 15). However, µmol/L estrogen are needed to inhibit LDL oxidation in vitro, a concentration at least a thousand-fold greater than that found physiologically. This would seem to imply that this nongenomic action is without biological implication. Shwaery et al. showed that physiologically relevant concentrations of E2 can have antioxidant effects on LDL. They found that LDL isolated from male plasma that had been incubated in vitro with 1 nmol/L E2 is protected from oxidation (7). Importantly, they demonstrated that LCAT esterification of E2 during the incubation with plasma was obligatory for LDL protection by E2. More recently, they showed that when estrone, which is not esterified by LCAT, is similarly incubated with plasma, the LDL is not protected from oxidation (16). Thus, minute amounts of the nonpolar E2 metabolites, E2-fatty acid esters (LE2),2 produced in blood by LCAT appear to have a pronounced antioxidative, cardioprotective function.
That E2 itself is an antioxidant is not surprising, as it
has a phenolic A ring similar to many other antioxidants, such as
vitamin E. However, fatty acid esterification of E2 at the
17ß-hydroxyl, a secondary alkyl hydroxyl, produces an antioxidant
with an unusual potency. Apparently, there are unknown chemical and
stereochemical constraints as well as enzymatic reactions that lead to
physiologically produced antioxidants. The extraordinary potency of the
estradiol ester metabolites as antioxidants led us to examine the
specificity of the D ring esterification of estrogens. We investigated
the site specificity of LCAT in comparison with that of
acyl-CoA:acyltransferase(s). We analyzed the products of esterification
by both LCAT in human blood and acyl-CoA:acyltransferase(s) in tissue
of four steroids: estriol (E3), E2, Adiol, and
testosterone (Fig. 1
). These studies show
a remarkable difference in the nature of the products formed by the two
enzymes. The results demonstrate the specificity of the esterification
of the estrogens and the potential of these unusual esters for
important physiological roles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human tissues, omental fat, placenta, and blood were obtained from patients undergoing surgery for reasons unrelated to these studies. These protocols were approved by the human investigation committee of Yale University School of Medicine (New Haven, CT).
Chromatography
Thin layer chromatography (TLC) was performed using 0.25-mm thick silica gel plates (Merck, EM Science, Darmstadt, Germany) with the following solvent systems: T1, hexane-ethyl acetate (7:3); T2, CHCl3-ethanol (9:1); and T3, benzene-ethyl acetate (85:15). Esters of E2 and E3 were eluted from the plates with CH2Cl2-CH3OH (9:1); Adiol and testosterone esters (TL) were eluted with benzene-acetone (4:1). In all cases the silica gel was placed into a glass wool-plugged Pasteur pipette and eluted with 1 mL of the appropriate solvent mixture. The solvent was evaporated under N2, and the resulting residue was partitioned between 1 mL diethyl ether and 0.2 mL H2O. The ether layer containing the steroid esters was evaporated and analyzed as described. The partition between ether and H2O was necessary to remove traces of silica to which the esters can reabsorb. The HPLC systems (Waters Corp., Marlborough, MA) were: 1) an M-6000A pump equipped with a U6K injector, a model 440 absorbance detector; and 2) a model 600E pump equipped with a U6K injector, a 484 absorbance detector, and a model 730 Data Module. HPLC columns and solvents on a LiChrosphere 100 Diol column (250 x 4.6 mm; Merck) used the following systems: system H1, CH2Cl2-isopropanol (23:2); system H2, isooctane-CH2Cl2 (1:4); system H3, isooctane/CH2Cl2 (7:3); system H4, isooctane/CH2Cl2 (2:3); system H5, isooctane/CH2Cl2 (4:1) on an Ultrasphere-Silica column (250 x 4.6 mm; Altex, Beckman Coulter, Inc., Berkeley, CA); and system H6, isooctane-isopropanol (49:1). All HPLC systems were run at a flow rate of 1.0 mL/min. The internal standards of E3 and E2 fatty acid esters were detected by the HPLC UV detector at 280 nm, TL were detected at 254 or 240 nm, and Adiol esters were detected at 210 nm.
Purification of 3H-labeled steroids
3H-labeled steroids were purified by chromatography to remove nonpolar impurities as follows. [3H]E2 was purified on a grade III alumina column (1 x 11 cm) equilibrated in CH2Cl2. The column was washed with 20 mL CH2Cl2, followed by 20 mL CHCl3. [3H]E2 was eluted using 20 mL CHCl3-ethanol (9:1). Fractions containing the product were combined and concentrated under flowing N2. [3H]Testosterone was purified on a grade II alumina column (1 x 11 cm) equilibrated in benzene. The column was washed with 10 mL benzene; 10 mL each of 5%, 10%, and 20% ethyl acetate in benzene; 0.1% ethanol benzene; and 20 mL 0.5% ethanol in benzene. Afterward [3H]testosterone was eluted with 20 mL 5% ethanol in benzene. Fractions containing the [3H]steroid were combined and evaporated under N2. [3H]E3 and [3H]DHEA were purified by HPLC, [3H]E3 in system H1 and [3H]DHEA in system H2.
[3H]Androstenediol synthesis
A solution of [3H]DHEA (46.64 µCi, 0.5 nmol) in MeOH (2 mL) was stirred for 5 h at room temperature with NaBH4 (10 mg, 0.264 mmol). The reaction mixture was poured into saturated aqueous NH4Cl (1.5 mL) and extracted with CH2Cl2 (three times, 1.5 mL), then with EtOAc (1.5 mL). The organic extracts were combined, dried over Na2SO4, and evaporated under N2 stream. The residue was dissolved in CH2Cl2 (80 µL) and isooctane (20 µL) and purified by HPLC in system H2. Fractions containing product [retention time (Rt) = 12 min] were combined and evaporated, yielding 4.1 x 107 cpm [3H]Adiol, a 79% yield.
Synthesis of steroid esters
E2-17ß-stearate (17), E3-16-oleate,
and E3-17ß-stearate were synthesized as described
previously (18). There is no special significance to the use of the
oleate ester (all of the different fatty acid esters behave similarly
in the chromatographic systems used in these studies).
Adiol-3ß-stearate was synthesized by esterification of
DHEA followed by reduction of the 17-ketone with
NaBH4 (19). Adiol-17ß-stearate was prepared by 1)
protection of the 3ß-hydroxyl group of DHEA as a
tert-butyldiphenylsilyl ether, 2) reduction of the 17-ketone to the
17ß-alcohol, 3) esterification with stearoyl chloride, and 4)
deprotection of the 3-hydroxyl group (19). Testosterone-17ß-stearate
was synthesized by direct esterification of testosterone with stearoyl
chloride in pyridine (20).
Saponification of E3 esters
Each sample was dissolved in 0.5 mL CH3OH, and 0.1
mL 1 N KOH was added. The mixture was incubated at 50 C
overnight. After incubation, the solution was neutralized with 1
N HCl. One milliliter of H2O was added, and the
CH3OH was removed under N2. The aqueous residue
was extracted twice with 5 mL ethyl acetate-ether (1:1). Controls were
treated similarly, except that H2O was added to the
incubation mixture instead of KOH. The organic extracts were combined
and evaporated under N2, and the residue was
chromatographed by TLC in system T2 using E3,
with E3-16-oleate and E3-17ß-stearate as
standards. Fractions migrating with the standards were eluted and
counted for radioactivity.
LCAT
Human blood was collected in heparinized tubes, and plasma was
obtained by centrifugation at 5,000 x g for 5 min.
Unless otherwise stated, approximately 500,000 dpm
(10-8 mol/L, final concentration) of the
3H-labeled steroid substrates were added to the incubation
tube, and the solvent was evaporated under N2. Afterward,
150 µL phosphate buffer, pH 7.4, and 50 µL plasma were added to the
tubes, and the mixture was incubated at 37 C for the indicated times.
In some experiments 1 mmol/L DTNB was included. The incubation mixtures
were then extracted as follows. For [3H]E2,
the enzymatic reaction was stopped by the addition of 0.8 mL
CH3OH containing 200 µg E2-17ß-stearate as
the internal standard. The reaction mixture was then extracted twice
with 2 mL hexane. The hydrocarbon layers were combined and dried under
N2, and the residue was purified by TLC in system
T1 [ratio to front (Rf) = 0.4].
E2 esters were visualized under long wave UV light after
spraying with primuline (21). The area containing the internal standard
was scraped from the plate, and the radioactivity was counted. For
[3H]E3, the reaction was stopped with 0.4 mL
CH3OH containing 200 µg each of
E3-16-oleate and E3-17ß-stearate as
internal standards. The reaction mixture was extracted twice, first
with 0.8 mL CHCl3 and then with 1.2 mL of the
CHCl3 layer from an equilibrated mixture of
CH3OH-CHCl3-H2O (1:2:1). The
CHCl3 layers were combined and evaporated, and the
resulting residue was partitioned between 2 mL each of
CH3OH-H2O (4:1) and isooctane-benzene (4:1).
The hydrocarbon layer was removed, and the aqueous CH3OH
layer was extracted again with 2 mL isooctane-benzene (4:1). The
hydrocarbon fractions contain the E3 esters, and they were
combined and evaporated. The E3 esters were purified either
by TLC as described for the E2 esters, but using system
T2 (Rf = 0.8), or by HPLC in system
H4. In these systems the 16- and 17ß-esters are not
separated. In some experiments, as indicated, after TLC, the 16- and
17ß-E3 esters were resolved by HPLC using system
H6. For [3H]testosterone, the enzymatic
reaction was stopped by the addition of 0.8 mL CH3OH. The
alcoholic mixture was extracted twice with 2 mL isooctane containing 50
µg testosterone-17ß-stearate. The hydrocarbon layers were
withdrawn, combined, and dried under N2. The residue was
purified by TLC in system T1 or by HPLC in system
H5. For [3H]Adiol, after the incubation, 200
µg each of the internal standards Adiol-3ß-stearate and
Adiol-17ß-stearate were added in CH3OH and extracted with
isooctane as described above. After evaporation of the hydrocarbon
layer under N2, the residue was purified by TLC in system
T3 (Rf = 0.3). The 17ß- and 3ß-esters were
not separated in this system. Adiol esters were visualized under UV
light after spraying with primuline. The fraction that comigrated with
the internal standards was eluted from the plate and partitioned
between water and diethyl ether, and the radioactivity in the ether
layer was counted. To resolve the 3ß- and 17ß-esters, the TLC
fraction was chromatographed by HPLC using system H3. The
yields in the incubations with E2, E3, and
testosterone were corrected for recovery of the internal standards as
determined in the UV of the HPLC. Adiol incubations were corrected for
the average recovery determined separately with radioactive Adiol
esters.
Human tissue Acyl-CoA:acyltransferase(s)
Microsomal fractions from human placenta and fat were prepared
as follows. The tissues were dissected free of extraneous matter and
washed thoroughly in ice-cold saline buffered with 10 mmol/L phosphate
buffer, pH 7.4, to remove blood. The tissue was minced, homogenized in
3 vol cold 0.25 mol/L sucrose solution with two separate 5-s bursts of
a Polytron homogenizer (Brinkmann Instruments, Inc.,
Westbury, NY) and then centrifuged at 700 x g for 10
min. The supernatant was centrifuged at 10,000 x g for
20 min, and the resulting supernatant was centrifuged at 105,000
x g for 60 min. The pellet was suspended in 0.1 mol/L
phosphate buffer, pH 7.4, and recentrifuged at 105,000 x
g for 60 min. The resulting pellet was suspended in the
phosphate buffer and stored at -70 C. 3H-Labeled
steroids were incubated with the acyl-CoA transferase in fat and
placental microsomes using procedures that we previously described (1).
The 3H-labeled steroids (500,000 dpm, 
10-8
mol/L, final concentration) were added to the incubation tubes in
alcoholic benzene and evaporated under N2. The microsomal
preparation (400 µg protein), 10 mmol/L ATP, 1 mmol/L CoA, 5 mmol/L
MgCl2, and 2 mmol/L dithiothreitol were added to the
incubation mixture in 200 µL (final volume) 0.1 mol/L phosphate
buffer. Incubations were carried out for 2 h at 37 C. The reaction
mixtures were extracted and analyzed as described above for the LCAT
experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E3.
[3H]E3 was incubated in human plasma
with or without 1 mmol/L DTNB for several time periods over 24 h
as indicated. The incubations were stopped at 0, 3, 6, 12, and 24
h, and aliquots were analyzed as described above. The residue obtained
after extraction of the incubation mixture was purified by TLC in
system T2. The area where the internal standards migrated
was eluted and purified by HPLC in system H4. The two
families of LE3, the 16- and 17ß-esters, were not
resolved in systems T2 or H4 (22). The
fractions from the HPLC containing the internal standards were counted
to quantify the formation of LE3. The results show the
time-dependent formation of a nonpolar metabolite that migrated with
the E3 ester standards (Fig. 2). As also shown in Fig. 2, the
formation of the esters was almost completely inhibited by the LCAT
inhibitor, DTNB.
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). LE3 synthesis was
determined by purification by TLC in system T2.
[3H]LE2 was determined by TLC in system
T1. In this experiment, the yield of
[3H]LE2 was about 2.3%, whereas the yield of
LE3 was 0.15%. As shown in Fig. 3, the formation of both
[3H]LE3 and [3H]LE2
was inhibited by 1 mmol/L DTNB.
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In another experiment, the nonpolar product
[3H]LE3, synthesized by incubation of
[3H]E3 with human plasma for 24 h, was
analyzed further to confirm its identification as LE3. The
hydrocarbon phase from the incubation extract was purified by column
chromatography with silica gel (see Materials and Methods).
The 3H-labeled metabolite was eluted from the column with
CHCl3 along with both internal standards
(E3-17ß-stearate and E3-16-oleate). After
evaporation, the residue was purified again by HPLC in system
H4. All of the [3H]LE3 eluted
from the column was coincident with the internal standards, at about 14
min. The [3H]LE3 was divided into two
fractions: one was saponified with KOH and the other, the control, was
incubated in parallel in aqueous alcohol. The reaction mixtures were
neutralized, extracted, and purified by TLC in system T2.
The fractions migrating with the standards of E3 and the
E3 esters were extracted and counted. As shown in Fig. 4, virtually all of the nonpolar
3H-labeled product, LE3, was hydrolyzed to
E3 by alkaline treatment. Conversely, almost none of the
[3H]LE3 was converted to E3 in
the control incubation with aqueous CH3OH. To confirm that
the saponified 3H-labeled material was E3, a
further reverse isotope dilution experiment was performed,
cocrystallization of the TLC purified 3H-labeled
saponification product with carrier E3. Approximately 600
dpm of the 3H-labeled fraction extracted from the
E3 area of the TLC plate was added to 20.5 mg
E3. The mixture was crystallized three times, and portions
of each of the crystals and the residue of the mother liquor were
weighed and counted. The results in Table 1 show that the specific activity of the
crystals and residues in the mother liquors remained constant
throughout each crystallization, confirming that the saponified
compound was [3H]E3. Thus, the nonpolar
product isolated from the incubation of
[3H]E3 with plasma migrates on HPLC with the
E3 esters and is converted by alkali into
[3H]E3. All of these characteristics are
distinctive of LE3.
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- and 17ß-esters
of LE3 (22). As shown in Fig. 5b, [3H]LE3 was
separated into two radioactive peaks in this system. Both peaks
comigrated with the two internal standards, either
E3-16-oleate or E3-17ß-stearate. In the
experiment shown, [3H]LE3 is composed of 78%
17ß-esters and 22% 16-esters. In three experiments performed
separately, the ratio of 17ß-esters to 16-esters of
LE3 was 3.2 ± 0.1. Thus, E3 is esterified
predominantly at the 17ß-hydroxyl group by LCAT. A summary of all of
the experiments is shown in Table 2.
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Although Adiol is known to be esterified by LCAT (4, 23, 24), the
specific product has not been characterized. To determine the nature of
the Adiol esters formed biosynthetically by LCAT,
[3H]Adiol was incubated overnight in human plasma with
and without 1 mmol/L DTNB. An aliquot of the residue obtained after
extraction of the incubation mixture was chromatographed by TLC in
system T3 (Rf = 0.3). Although the C-3 ester
migrates slightly faster than the C-17 ester in this system, they are
not cleanly separated. The area of the plate containing both standards
was extracted as described above, and an aliquot of the residue was
counted. In three separate experiments, the yield of esterification
ranged between 5.1–8.6%. In the incubations with DTNB, the yield was
0.1%. The remainder of the extract was analyzed by HPLC in system
H3, which separates the Adiol 3ß- and 17ß-esters
(Rt = 13 and 16 min, respectively). As shown in Fig. 5d
, all of the radioactivity migrated with the internal standard,
Adiol-3ß-stearate. No [3H]17ß-ester was observed. The
same results were obtained in three separate experiments. Thus, the
[3H]Adiol fatty acid esters formed by LCAT are
exclusively Adiol-3ß-fatty acid esters. Although Adiol is a substrate
for LCAT, unlike E2 and E3, it is not
esterified at the 17ß-hydroxyl group.
Testosterone
The [3H]C-19 steroid was incubated in human plasma
with or without 1 mmol/L DTNB and extracted as described above. The
residue obtained from the extract of the incubation mixture was
analyzed by HPLC in system H5 (Rt = 14 min).
Aliquots of each fraction were counted. No radioactivity was found in
any of the fractions (see Fig. 5c). Thus, despite having an
enzymatically accessible 17ß-hydroxyl group, testosterone is not
esterified by LCAT.
Acyl-CoA:acyltransferase(s)
The four 3H-labeled steroidal substrates,
E2, E3, testosterone, and Adiol, were incubated
with the microsomal fraction from human placenta and fat with an
acyl-CoA-generating system as described above. After incubation for
2 h at 37 C, the reaction mixtures were extracted and purified
exactly as described for the LCAT experiments (representative results
are shown in Fig. 5, e–h). In general, the rate of esterification of
the steroids was lower in placenta than in fat. In three separate
experiments with different placental preparations, the yields were: for
E2, from 0.25–0.5%; for E3, 0.1–0.15%; for
testosterone, 0.15–0.2%; and for Adiol, 1–4%. The esters of
E3 and Adiol formed in the placenta were characterized as
described in the LCAT experiments. For Adiol, the ratio of 17ß- to
3ß-esters (±SD) was 1.1 ± 0.2. For E3,
the ratio of 17ß- to 16-esters was 0.94 ± 0.04. In three
separate experiments with different preparations of omental fat, the
yields were: for E2, from 7.8–10% (Fig. 5e); for
E3, from 1.2–2.5%; for testosterone, from 2–3.6% (Fig. 5g); and for Adiol, 26–31%. The esters of E3 and Adiol
formed in the fat were characterized. For E3, the ratio
(±SD) of 17ß- to 16-esters was 1.09 ± 0.02
(Fig. 5f). For Adiol, the ratio (±SD) of 17ß- to
3ß-esters was 2.8 ± 0.1 (Fig. 5h). The results are summarized
in Table 2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-steroids as well as
E2 was reported by Jones and James in 1985 (4). Although
many different steroids are esterified in tissues by
acyl-CoA:acyltransferase(s) enzymes (25), LCAT has a more limited
capability. Most steroids that are esterified in tissues are not
esterified in blood. For example, several C-19 steroids with
17ß-hydroxyl groups, such as testosterone (4) and
5-dihydrotestosterone, are not esterified by LCAT (4, 23, 24). In
addition, steroids containing a 3-hydroxyl group, such as
androsterone, are also not esterified by LCAT (26). This high substrate
specificity makes it especially surprising that E2 is
esterified by LCAT, because other steroids with structurally similar D
rings are not esterified. For example, as shown in Fig. 5c, under
conditions where E2 is readily esterified by LCAT at C-17,
testosterone is not esterified despite also having a 17ß-hydroxyl
group. Yet both steroids are esterified in placenta (results above) and
fat. Human fat was chosen for these experiments because it seemed to be
the most likely tissue to contain the highest steroid acyltransferase
activity. We have previously shown that human fat has the highest
concentration of LE2 (27), and that in male rats, fat and
testes are the only tissues that contain TL (20). As expected, placenta
had much lower levels of acyltransferase activity than fat. When LCAT esterification of E2 was first discovered, it was assumed that E2 was esterified at C-17 because the D ring hydroxyl had been shown to be the site of esterification in tissues (17), and because estrone, which does not have a 17-hydroxyl group, is not esterified by either LCAT (4) or acyl-CoA:acyltransferase(s) (28). Later, we isolated and characterized endogenous LCAT-synthesized LE2 from human ovarian follicular fluid and showed that it consisted solely of 17ß-fatty acid esters of E2 (5). LE2 in ovarian follicular fluid is synthesized by LCAT (6).
The esterification of Adiol is especially interesting because, unlike other C-19 steroids such as testosterone, it is a known substrate for LCAT (4, 23, 24). This C-19 steroid has received considerable interest because, contrary to what might be expected from its structure, it is an unusual estrogen (29, 30, 31). Adiol has two secondary hydroxyl groups, one at 3ß and the other at 17ß, both of which are potentially esterifiable. There have been several studies of the esterification of Adiol by tissue acyl-CoA:acyl transferase. It has been shown that both the 3ß- and 17ß-fatty acid esters are synthesized. In human breast cancer microsomes, approximately equal amounts of the two esters are produced (32). In studies with breast cancer cell lines, both esters of Adiol have also been identified. In one of these studies, it was reported that the 17ß-esters predominated (33), whereas in another, the 3ß-esters were the major product (34). In the present studies with human placental microsomes, the two families of esters were produced in approximately equal amounts, with slightly more of the 17ß-esters formed. In fat, the 17ß-esters were produced in a greater than 3:1 ratio. The difference in the rate of esterification of the two sites by placenta and fat may indicate that different acyl-CoA:acyltransferase(s) esterify the two hydroxyls and that the concentrations of the two enzymes are different in these tissues. Nevertheless, in both tissues the 17ß-hydroxyl group is efficiently esterified.
Although Adiol is known to be esterified by LCAT, the nature of the
product has not been determined previously. We characterized by HPLC
the esters of Adiol that were formed in plasma. As shown in Fig. 5d, large amounts of the C-3 esters were detected, but no 17ß-esters were
found. Thus, unlike testosterone, Adiol is an excellent LCAT substrate,
but it, too, is not esterified at C-17. The comparison of LCAT
esterification to that of acyl-CoA:acyltransferase(s) of the C-19
steroids is especially remarkable because, in contrast to LCAT, the
acyl-CoA:acyltransferase(s) in tissues esterifies the 17ß-hydroxyl of
both testosterone and Adiol.
E3 is a poor substrate for LCAT. It is esterified at less
than 10% the rate of E2. Nevertheless, the very fact that
E3 is esterified at all is surprising, because it is a very
polar steroid. Shwaery et al. (16) also noted small amounts
of a nonpolar metabolite formed in the incubation of E3
with plasma, but the product was not characterized. E3 is
not only far more polar than the major LCAT substrate, cholesterol, but
it is also more polar than most estrogens and their metabolites. This
is shown by the fact that E3 can be easily separated from
most other aromatic steroids by a simple partition between benzene and
an aqueous buffer (35). With the exception of E3, this
procedure extracts almost all of the estrogens into the hydrocarbon
phase. The formation of the nonpolar E3 metabolite in
plasma is enzymatic; it increases with time, and it is inhibited by
DTNB. As would be expected for LE3, the nonpolar product is
saponified into E3, which was identified by both its
chromatographic properties as well as cocrystallization with carrier
E3. LCAT-synthesized LE3 was characterized by
HPLC, in a system that resolves the 16- and 17ß-esters. As shown
in Fig. 5b, there was considerably more of the 17ß-ester than the
16-ester formed by LCAT (>3:1 respectively). In contrast to LCAT,
the acyl-CoA:acyltransferase(s) in both fat and placenta (see
Results) synthesizes about equal amounts of the two families
of esters. We have previously found in another model system, rat lung
microsomes, that the 16-hydroxyl is esterified in slightly greater
amounts than the 17ß-hydroxyl (22). The prevalence of the
17ß-esters in the LCAT esterification of E3 is
surprising, because although the enzyme can obviously esterify both D
ring hydroxyls, the 16-hydroxyl is sterically much more accessible.
The 17ß-hydroxyl is shielded by the C-18 methyl group, which makes
esterification at this site energetically much more difficult than that
at the 16-hydroxyl group.
5-3ß-Hydroxysteroid fatty acid esters produced
in situ by LCAT in blood are bound exclusively to
lipoproteins (36, 37, 38). E2-fatty acid esters also bind only
to lipoproteins and to no other proteins in blood (39). Bélanger
et al. found that steroid esters, unlike cholesterol esters,
do not require cholesterol ester transfer protein for transport from
high density lipoprotein, where they are synthesized to LDL (40).
Furthermore, they showed that lipoprotein-bound steroid esters can be
taken up into cells by lipoprotein receptors where they are hydrolyzed
into the free steroid (41, 42, 43). In this manner, circulating steroid
esters can act as a reservoir of the free steroids and, thereby, a
source of biologically active hormones.
The recent finding that LCAT-synthesized LE2 acts as an
extremely potent antioxidant points to an important physiological role
for these E2-esters (7, 16). It is known that
LE2 circulates in human female blood (27, 44), albeit in
low concentrations (10 pmol/L). The low concentration, in addition
to analytical complexities, have made accurate quantification
difficult. Consequently, there is little known about the physiological
control of circulating LE2. It appears that, as would be
expected, there is a correlation between E2 and
LE2 in blood (27), and that the fatty acids comprising
LE2, both biosynthetic (6) and endogenous (5), are
predominantly unsaturated. However, there are no rigorous studies of
LE2 levels or fatty acid composition in premenopausal or
menopausal women, nor is anything currently known about the
relationship of endogenous LE2 to arteriosclerosis or any
other pathological condition. These questions become important in light
of the finding that E2 esterification leads to a potent
antioxidant protection of LDL, as this nongenomic action may be a major
factor in the well known cardiovascular protection caused by estrogens.
The present studies demonstrate that there is both stringent substrate
and regio- specificity for LCAT esterification; only
estrogens, not structurally similar C-19 steroids, are esterified in
the D ring. The esterification of the sterically hindered
17ß-hydroxyl group of E3, as in E2, may
illustrate a previously unrecognized steric requirement that leads to
extraordinarily potent antioxidants.
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Acknowledgments |
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Footnotes |
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2 L as a prefix or suffix as in LE2,
LE3, TL, or Adiol L, indicates the phrase "lipoidal
derivative" (fatty acid esters) of estradiol, estriol, testosterone,
and 5-androstenediol, respectively.
Received January 19, 1999.
Revised March 2, 1999.
Accepted March 3, 1999.
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5-androstene-3ß,17ß-diol with
estradiol and dihydrotestosterone receptors in human myometrial and
mammary cancer tissue. J Clin Endocrinol Metab. 40:373–379.5 adrenal steroids. Endocrinology. 127:2757–2762.5-steroids to long-chain fatty
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G. Serviddio, G. Loverro, M. Vicino, F. Prigigallo, I. Grattagliano, E. Altomare, and G. Vendemiale Modulation of Endometrial Redox Balance during the Menstrual Cycle: Relation with Sex Hormones J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2843 - 2848. [Abstract] [Full Text] [PDF]
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H. Helisten, A. Höckerstedt, K. Wähälä, A. Tiitinen, H. Adlercreutz, M. Jauhiainen, and M. J. Tikkanen Accumulation of High-Density Lipoprotein-Derived Estradiol-17{beta} Fatty Acid Esters in Low-Density Lipoprotein Particles J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1294 - 1300. [Abstract] [Full Text]
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