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Original Studies |
Departments of Medicine (H.H., A.H., M.J.T.), Organic Chemistry (K.W.), and Obstetrics and Gynecology (A.T.), Division of Clinical Chemistry (H.A.), University of Helsinki, 00014 Helsinki; Folkhälsan Research Center (H.A.), Institute for Preventive Medicine, Nutrition, and Cancer, 00280 Helsinki; and Department of Biochemistry (M.J.), National Public Health Institute, FIN-00300 Helsinki, Finland
Address correspondence and requests for reprints to: Matti J. Tikkanen, M.D., Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland. E-mail: matti.j.tikkanen{at}helsinki.fi
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were interested in yet another cardioprotective mechanism based on the antioxidant efficacy of estrogen. Many estrogenic substances (6, 7, 8), including phytoestrogens (9), are known to be powerful antioxidants in lipid-aqueous systems, for example inhibiting LDL peroxidation in vitro. The antioxidative potential of human estrogens seems to depend on the presence of a free hydroxyl group in the aromatic ring A of the steroid molecule (10, 11, 12, 13). Reports indicating that endogenous human estrogens could be rendered fat-soluble by esterification with fatty acids in vivo, and the subsequent detection of such esters in blood and fat (14, 15), suggested a possible mechanism by which they could protect LDL against oxidation. Esterified estrogens can become associated with lipoproteins in vitro (16, 17, 18, 19, 20, 21) and with rat lipoproteins in vivo (16), indicating the possibility that estrogen derivatives may become incorporated into human lipoprotein structure in vivo (17, 19). This could offer an explanation for several reports claiming increased oxidation resistance of LDL in association with estrogen administration (22, 23, 24, 25). In the present study, we investigated possible mechanisms by which LDL molecules might incorporate estrogen to improve their oxidation resistance. We started with human follicular fluid, which is a rich source of estrogen and is reported to contain only one lipoprotein class, HDL (26, 27, 28, 29, 30, 31). In addition, experiments were carried out using human plasma. Our results demonstrated that lechitin:cholesterol acyl transferase (LCAT)-mediated esterification of 17ß-estradiol (17ß-E2) occurred in HDL particles and they further suggested a transfer mechanism of estradiol esters from HDL to LDL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We obtained fresh follicular fluid from aspirates of women undergoing oocyte retrieval for in vitro fertilization at the Helsinki University Central Hospital. The study was approved by the Ethics Committee of the Department of Obstetrics and Gynecology, and written informed consent was obtained from patients. Ovarian stimulation was induced by combining GnRH analog with gonadotrophins. Follicular fluid was centrifuged twice (2300 x g, 15 min, 10 C) to remove blood cells and granulosa cells as well as nonspecific cell debris. Blood was drawn from the same women as well as from healthy, normolipidemic male donors (aged, 21–25) who were taking no medication, into EDTA-containing vacuum tubes. Plasma was prepared by immediate centrifugation at 2500 x g, 15 min, 4 C and was used for experiments during the same day.
Incubation of 17ß-E2 in the presence of follicular fluid and plasma
Labeled estrogen, [2, 4, 6, 7-3H(N)] 17ß-E2 (New England Nuclear, Boston, MA; specific activity 72 Ci/mmol), in 0.5 M HEPES buffer (pH 7.4) was added to follicular fluid to reach a final concentration of 2 x 106 cpm/mL (corresponding to 52 nmol/L E2) in a total volume of 4 mL. The mixture was incubated for 24 h at 37 C in the absence and presence of 1.5 mmol/L dithionitrobenzoic acid (DTNB) (Sigma, St. Louis, MO). Plasma incubations were performed identically during the same experimental set.
Isolation of lipoproteins
Lipoproteins were isolated by sequential ultracentrifugation (32) using a Beckman Optima LE-80K ultracentrifuge and a Ti 50.4 rotor as: very low density lipoprotein at density d < 1.006 g/mL (270,000 x g, 3.5 h, 10 C), LDL in the density range 1.006–1.063 g/mL (160,000 x g, 17 h, 10 C), and HDL in the density range 1.063–1.21 g/mL (270,000 x g, 24 h, 10 C). In follicular fluid lipoprotein was recovered only in the HDL density range according to a protocol applied for plasma samples. In some experiments, HDL was recentrifuged ("washed") at the upper density 1.21 g/mL to remove any weakly associated proteins.
Purification of lipoproteins by size-exclusion chromatography
Ultracentrifugally isolated HDL from follicular fluid as well as
LDL and HDL isolated from plasma were further purified by gel
filtration on Sephadex G-25 (column dimensions, 2 x 20 cm or
1 x 20 cm; Pharmacia Biotech, Uppsala, Sweden). The
applied sample volume was 2.5 mL. Phosphate-buffered saline (PBS), pH
7.4, was used as elution buffer. Lipoproteins eluted in the column void
volume (Vo) (Figs. 2A
and 3, A and C). Radioactivity in the
elution fractions was determined by liquid scintillation counting
(Rack-beta; Wallac, Turku, Finland) with a 3H
counting efficiency of 51.1%. The fractions containing lipoproteins
(LDL or HDL, depending on the experiment) were pooled for further
analysis.
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The pooled plasma or follicular fluid lipoprotein fractions (LDL
or HDL) obtained by gel filtration on Sephadex G25 were extracted four
times with ethylacetate/diethylether (1:1, v/v) (2.5 x sample
volume). The water phase was quick frozen, followed by removal of the
organic layer and its evaporation to dryness under N2. The
dry residues were dissolved in 0.3 mL hexane/chloroform (1:1, v/v). To
separate esterified 17ß-E2 from the free
17ß-E2, samples (in 0.6 mL hexane/chloroform, 1:1, v/v)
were applied on a Sephadex LH-20 column (0.5 x 5 cm;
Pharmacia Biotech (33) and eluted with 10 mL
of the same solvent at room temperature collecting 1-mL fractions.
Esterified 17ß-E2 was eluted first, after which the
elution was continued with methanol to elute the unesterified
17ß-E2 (see Figs. 2B
and 3, B and D). All fractions were
evaporated to dryness under N2 and dissolved in 0.5 mL
methanol. The radioactivity was counted in each fraction.
For identification of saponified steroids, Sephadex LH-20 chromatography was carried out using 9% toluene in methanol, which separates unesterified estrone, 17ß-E2 and estriol from each other (34). Saponification was carried out by dissolving the sample in 1 mL 1n KOH in methanol and incubating for 2 h at 60 C. The mixture was neutralized by HCl, and the methanol was removed under N2.
Thin-layer chromatography
Samples (approximately 1000 cpm) obtained from the "ester" and "free" E2 fractions after chromatography on Sephadex LH-20 were applied to TLC plates (20 x 20 cm, Silica gel 60; Merck, Germany). The following nonradioactive standards were used: 17ß-E2 (Steraloids Inc.), 17ß-E2 stearate (Steraloids Inc., and E2-3, 17ß-dioleate. In short, E2-3, 17ß-dioleate was obtained by stirring 17ß-E2, dimethylaminopyridine, and oleyl chloride in pyridine at 60 C under argon atmosphere. After 2 h, the mixture was poured into water, acidified, and extracted with ether. Purification by flash chromatography gave an oily product in 62% yield. E2-3, 17ß-dioleate, one spot on TLC, was characterized by 1H and 13C NMR. Ethylacetate/hexane (1:10, v/v) was used to develop the plates. The location of the standards was determined by visualization under ultraviolet light after rhodamine staining. The Rf-values of the standards were: 17ß-E2, 0.07; E2-17ß-stearate, 0.27; and E2-3, 17ß-dioleate, 0.73.
Transfer of [3H] estradiol-17ß between lipoproteins
Plasma was prepared from freshly drawn blood obtained from normolipidemic males. LDL fractions were purified by gel filtration as described under Purification of lipoproteins. This purified, nonradioactive LDL was incubated with HDL isolated from the same subject, which contained [3H]E2 esters (prepared by incubation with plasma as described above). Incubations were carried out in 3 mL PBS. For each incubation HDL corresponding to 12,500 cpm (HDL protein between 0.3 and 0.9 mg) was used. The amount of LDL (defined as LDL protein) used in each incubation was half of the HDL protein used (i.e. 0.15–0.45 mg) in the same incubation. The incubations were carried out in the absence as well as in the presence of various amounts of cholesterol ester transfer protein (CETP) at 37 C for 3, 6, 12, and 24 h. After incubations, lipoproteins were reisolated by ultracentrifugation as described under Isolation of lipoproteins. The transfer of [3H]E2-17ß esters from HDL to LDL was measured by determining the radioactivity in both donor (HDL) and acceptor (LDL) lipoproteins.
Western blot analysis
For Western blotting, the HDL proteins were resolved by 12.5% homogeneous SDS-PAGE under reducing conditions (35) and electrotransferred to Hybond-C (Amersham Corp., Arlington Heights, IL) or nitrocellulose membranes. Electrotransferred proteins were analyzed using the method of Towbin et al. (36). To visualize human CETP, a rabbit polyclonal antibody raised against a synthetic peptide with identity to amino acids 335–349 of human CETP was used in dilution of 1:1000. Goat antirabbit IgG conjugated to horseradish peroxidase was used as a secondary antibody (final dilution, 1:2000). Antigen-antibody complexes were visualized by using a chromogenic peroxidase substrate. Human apoA-I and LCAT proteins were probed with a rabbit polyclonal antibody (apoA-I) and a monoclonal antibody 2H11 (LCAT), that were a kind gift from Dr. Yves Marcel (University of Ottawa Heart Institute, Ottawa, Canada). For detection of apoA-I, peroxidase-conjugated goat antirabbit antibody was used whereas the bound monoclonal antibody in LCAT analysis was detected using horseradish peroxidase-conjugated goat antimouse IgG (Nordic Immunology, The Netherlands) and a chromogenic peroxidase substrate.
Purification and assay of CETP
Purification of CETP from human plasma was performed essentially as described recently (37, 38). CETP activity was determined with a radiometric method in which CETP transfers radiolabeled cholesteryl esters from LDL to HDL (39). Briefly, plasmapheresis plasma was ultracentrifuged at density of 1.21 g/mL and the bottom fraction (d > 1.21 g/mL) was applied to a hydrophobic Butyl-Toyopearl 650 (M) column and run essentially, as described by Ohnishi et al. (40). CETP-active fractions were pooled and directly applied to a heparin-Sepharose CL 4B column (1.5 x 8.5 cm; Pharmacia) equilibrated with 25 mmol/L Tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4) containing 50 mmol/L NaCl and 1 mmol/L EDTA. During this purification step, plasma phospholipid transfer protein binds to the heparin column, whereas CETP is eluting in the nonbound fraction. The unbound fraction expressing CETP activity was dialyzed against 25 mmol/L Tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4) containing 1 mmol/L EDTA buffer that was used to equilibrate a Mono Q anion-exchange column. After dialysis, the sample was loaded on to a Mono Q HR 5/5 column (Pharmacia) and run at room temperature using a Merck-Hitachi high-performance liquid chromatography system. Elution was performed with a linear NaCl gradient (0–0.5 mol/L) prepared in equilibration buffer. The fractions were analyzed for CETP activity, and the most active fractions eluted in the NaCl range of 80–110 mmol/L. The CETP-active fractions were stored at -70 C until use. The activity of CETP in different purified batches varied between 15 and 25 nmol cholesteryl esters transferred per h/mL. Purified CETP did not contain PLTP, LCAT, lipoprotein lipase, or hepatic lipase activity.
Other methods
The concentrations of apoA-I and apoB-100 were measured by an immunochemical assay (Orion Diagnostica, Finland), and concentrations of cholesterol (Boehringer Mannheim, Mannheim, Germany), triglycerides (Roche, Switzerland), and phospholipids (Wako Chemicals, Germany) by enzymatic tests. Protein levels were determined by the method of Lowry et al. (41). Concentration of unesterified E2 was measured by RIA (Sorin Diagnostics, Italy). Statistical analyses were performed by Student’s t test.
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Results |
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In follicular fluid, obtained from women undergoing gonadotrophin
stimulation, the mean total cholesterol concentration was 0.95 ±
0.14 mmol/L, whereas the total cholesterol concentration in homologous
plasma was 4.47 ± 0.25 mmol/L (n = 5). The major
apolipoprotein, apoB-100, was not detectable in follicular fluid,
whereas apoA-I was present (Fig. 1, lane
3) but its concentration was lower than
in plasma (0.84 ± 0.11 vs. 1.62 ± 0.15 mg/mL,
respectively). The presence of LCAT was also detected by immunoblotting
(Fig. 1, lane 3). The HDL cholesterol concentration in follicular fluid
was 0.61 ± 0.21 mmol/L, and that in homologous plasma was
1.30 ± 0.15 mmol/L. The composition of follicular fluid HDL was
characterized by a lower phospholipid mass percentage and a higher
triglyceride mass percentage value when compared to plasma HDL (Table 1). Concentrations of
17ß-E2 in follicular fluid ranged between 900 and 2000
nmol/L.
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Incubation of [3H]E2-17ß in the
presence of follicular fluid. Follicular fluid was incubated with
[3H]E2-17ß with and without DTNB (1.5
mmol/L final concentration), followed by isolation of HDL by
ultracentrifugation. This HDL fraction was gel-filtered on Sephadex
G-25. Figure 2A shows that the
radioactivity coeluted with the protein peak in the void volume
(Vo), suggesting that it was associated with HDL. When the
same incubation was carried out in the presence of the LCAT inhibitor
DTNB, almost no radioactivity could be detected in the elution position
of HDL (Fig. 2A), suggesting that esterification was a prerequisite for
incorporation of [3H]E2-17ß into HDL.
Following extraction of the HDLcontaining fractions (elution
volume range, 12–15 mL) with hexane/chloroform and subsequent
hydrophobic chromatography on Sephadex LH-20, a major part of the
radioactivity (82.8%) was eluted in the "ester fraction"
(fractions 1–3) and only 9.0% was detected in the "free fraction"
(fraction 12) (Fig. 2B). There was no detectable radioactivity in the
"ester" fraction of the sample that was incubated in the presence
of DTNB. The "ester fractions" were further analyzed by TLC that
offered further evidence that most of the radioactivity (78%) migrated
in the same position as the ester standard
(E2-17ß-stearate, Rf-value 0.27) (Table 2).
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Incubation of [3H]E2 in the presence of
plasma. Following incubation of
[3H]E2-17ß with normolipidemic male plasma
and isolation of LDL and HDL by ultracentrifugation, these lipoproteins
were gel filtered on Sephadex G25 (Fig. 3, A and
C). Both lipoprotein fractions were then
subjected to gel chromatography on Sephadex LH-20 (Fig. 3, B and D). On
gel filtration, the radioactivity coincided with the protein
(apolipoprotein) peaks, suggesting that the
[3H]E2-17ß was attached to the
lipoproteins. Further hydrophobic chromatography using Sephadex LH-20
demonstrated that most of the radioactivity was recovered in the
"ester fraction" (Fig. 3, B and D). Practically no radioactivity
was detected in the "ester" fractions when DTNB was added to plasma
prior to the incubations.
Incubation of native LDL with [3H]estradiol-17ß- labeled HDL
Because it is presumed that LCAT is associated exclusively with
HDL, we explored the possibility that
[3H]E2-17ß-esters formed in HDL could be
transferred to LDL by means of a CETP-catalyzed process explaining the
presence of esterified [3H]E2-17ß in LDL.
Nonradioactive (native) LDL was incubated with HDL (12,500 cpm)
obtained from plasma incubations with
[3H]E2-17ß in the absence and presence of
CETP. As shown in Fig. 4A, during incubation without adding CETP the radioactivity in LDL
increased proportionally with time, while subsequently the
radioactivity in HDL decreased. In the presence of purified CETP (final
cholesteryl ester transfer activity per incubation, 8 nmol cholesteryl
ester transferred/h) this shift in radioactivity from HDL to LDL was
significantly accelerated (Fig. 4B). The transfer of label from HDL
(donor) to LDL (acceptor) in multiple incubations was quantitated by
determining the LDL/HDL (cpm/cpm) ratio at various time points. The
significance of the difference between the mean LDL/HDL count ratios at
each time point was analyzed by paired t test (mean and
SD): 3 h (0.23 ± 0.052 vs. 0.34
± 0.060, n = 4), P = 0.028 without and with CETP.
The corresponding values for the other time points were: 6 h
(0.40 ± 0.169 vs. 0.55 ± 0.149, n = 5),
P = 0.032; 12 h (0.46 ± 0.090 vs.
0.76 ± 0.192, n = 3), P = 0.035; 24 h
(0.82 ± 0.210 vs. 0.90 ± 0.264, n = 5),
P = 0.388.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incubation of 3H-labeled 17ß-E2 with human follicular fluid produced a HDL-bound 3H-labeled substance, most of which coeluted with 17ß-E2 ester standards on a hydrophobic chromatography. These findings suggested that LCAT-mediated production of fat-soluble fatty acid esters of 17ß-E2 was a prerequisite for lipoprotein incorporation of this steroid. Although the 3H-label coeluted with synthetic ester standards in the "ester" fraction on Sephadex LH-20 and was abolished by LCAT inhibition, we sought to further confirm that it was identical to 17ß-E2 ester and not some other lipophilic derivative. Thus, we demonstrated that the 3H-label obtained from the "ester" fraction and synthetic E2-17ß stearate comigrated during TLC analysis. Moreover, saponification of the "ester" fraction resulted in a shift of the 3H label from the "ester" to the "free" fraction. We also showed that estrone (E1) or estriol (E3) were not present in the "ester" fraction. Conversion of E2-17ß to E1 and E3 was theoretically possible because the necessary 17ß-hydroxysteroid dehydrogenase enzymes present in ovarian granulosa cells (43) could have contaminated the follicular fluid when it was collected, and following esterification, E1 and E2 could have been incorporated into HDL. These initial experiments strongly suggested that LCAT-mediated esterification of E2-17ß had occurred, and that the ester form could be incorporated into at least one lipoprotein, follicular fluid HDL, in line with a previous report (44).
After the experiments with follicular fluid we wanted to know whether a similar esterification and incorporation into HDL occurred in plasma, and if so, how the esterified 17ß-E2 might be transported from HDL to LDL particles. Following incubation of 3H-labeled 17ß-E2 with plasma, ultracentrifugal isolation of the HDL, its purification by gel filtration and hydrophobic chromatography, the tritium label was detectable almost exclusively in the "ester" fraction. The addition of the LCAT inhibitor DTNB to the plasma incubation almost completely abolished the lipoprotein-bound radioactivity. Analogous findings were observed when the LDL fraction was isolated and processed in the same way: 3H-label was recovered in the "ester" fraction, except when DTNB had been added to the plasma incubation.
These findings raised the question about mechanisms that could explain how these lipophilic estrogen esters, apparently produced in HDL, were transported to LDL particles. We hypothesized that CETP would also be responsible for this transport of 17ß-E2 esters between lipoproteins. This was considered possible as cholesterol and 17ß-E2 share a somewhat similar ring structure. It was demonstrated that coincubation of native LDL with HDL containing 3H-labeled 17ß-E2 ester in a buffer solution resulted in transfer of the label to LDL and that this transfer was significantly accelerated by addition of purified CETP to the incubation mixture. Conversely, "washing" of the HDL fraction by a second ultracentrifugation reduced the spontaneous transfer, presumably due to reduction of the CETP adhered to HDL particles. The present study provides for the first time in vitro evidence that CETP participates in the transfer of E2 esters. It is generally accepted that CETP has specificity for both neutral lipids (cholesterol esters and triglycerides) and phospholipids (45). However, the observation on the CETPfacilitated acceleration of the transfer of 17ß-E2 esters from HDL to LDL is relevant in the context of the recent data reporting on the CETP mechanism. A new model of the transfer reaction mechanism to CETP was constructed based on the crystal structure of human bactericidal permeability increasing protein. Both of these proteins belong to the lipopolysaccharide binding/lipid transfer protein family (46). These proteins share structural homology and are suggested to have a boomerang-shaped structure with two domains (N- and C-terminal), both of which contain a nonpolar lipid-binding pocket (47). The model suggested that a hydrophobic pocket in the amino-terminal domain of CETP may have a role in neutral lipid binding and transfer (46, 48). Furthermore, the N-terminal active site may be involved in determining the substrate species specificity for the transfer (49) and, accordingly, the transfer of E2 esters might be mediated via this site. The role of CETP was recently evaluated as a possible factor in transfer of neutral steroid esters. Provost et al. (50) claimed that the fatty acid esters of dehydroepiandrosterone and pregnenolone were transferred from HDL to very low density lipoprotein and LDL by a mechanism not dependent of CETP. However, this observation does not contradict the possibility that CETP mediates estradiol ester transfer, but suggests that dehydroepiandrosterone and pregnenolone esters have very weak or null affinity to the lipid-binding pocket of CETP. Additional studies are needed to elucidate a detailed CETP-mediated transfer of E2 esters. Thus, the present data show that CETP is able, at least in vitro, to transfer esterified 17ß-E2 from HDL to LDL particles. There is currently methodologically no easy way to clarify in what position the 17ß-E2 esters are located in the lipoprotein structure. One possibility is that they are aligned on the lipoprotein surface in the same way as phospholipids, the fatty acid chains directed toward the core and the ring nucleus with the hydrophilic A-ring hydroxyl group on the surface of the lipoprotein, thus providing functionally essential conformation in the antioxidant process.
In summary, we set out to explore the processes that could result in incorporation of estrogens in LDL structure, a theoretical mechanism by which estrogens could render LDL particles less susceptible to oxidative modification. The present in vitro data demonstrate that enzyme/lipophilic transfer systems in human follicular fluid and plasma were capable of incorporating [3H]E2-17ß into HDL after conversion to fatty acid ester, and that [3H]E2-17ß ester present in HDL could be transported in plasma to LDL particles in a process that were, at least in part, catalyzed by CETP.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
Received July 27, 2000.
Revised November 13, 2000.
Revised December 5, 2000.
Accepted December 5, 2000.
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