© 1997 American Heart Association, Inc.
Articles |
Effects of Contraceptive Estrogen and Progestin on the Atherogenic Potential of Plasma LDLs in Cynomolgus Monkeys
From the Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC.
Correspondence to Dr John S. Parks, Department of Comparative Medicine, Bowman Gray School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157. E-mail jparks{at}bgsm.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract This study was designed to determine the effect of oral contraceptive treatment (estrogen and progestin), alone or in combination, on LDL composition and atherogenic potential in cynomolgus monkeys fed an atherogenic diet. Groups (n=8 each) of monkeys were untreated (control) or treated with ethinyl estradiol (EE), levonorgestrel (LNG), or triphasic oral contraceptive (EE+LNG) for 1.5 years before plasma LDLs were isolated for characterization. Total plasma cholesterol concentrations were unaffected by the treatments. LDL particle size (measured as LDL molecular weight, g/µmol) was significantly smaller in the EE (4.61±0.09) and EE+LNG (4.43±0.09) treatment groups compared with the control (4.99±0.09) or LNG (5.29±0.17) groups and contained fewer molecules of free and esterified cholesterol. Both the EE and EE+LNG groups had significantly less cholesterol and apolipoprotein B distributed in the d=1.015 to 1.025 g/mL subfraction and correspondingly more in the d=1.025 to 1.035 g/mL subfraction of LDL compared with the control and LNG groups. The apolipoprotein E content (molecules/particle) of LDL was significantly less in the EE (0.35±0.1) and EE+LNG (0.28±0.1) groups compared with the control (0.86±0.2) and LNG (0.99±0.2) groups, and this trend was apparent in all three LDL subfractions. The atherogenic potential of LDL was tested using an in vitro binding assay to arterial proteoglycans. Twice as much LDL bound to arterial proteoglycans in the LNG group (11.3±1.8% of total LDL cholesterol in the incubation) compared with the control (6.4±1.9%), EE (5.5±1.5%), or EE+LNG (5.2±1.2%) groups. We conclude that EE and EE+LNG treatment alters the composition of LDL toward a less atherogenic particle that is smaller and more dense, contains less cholesterol and less apolipoprotein E, and is less reactive with arterial proteoglycans compared with LNG treatment. The inclusion of EE in the triphasic oral contraceptive treatment was sufficient to negate the potentially atherogenic effects of LNG on LDL composition. (Arterioscler Thromb Vasc Biol. 1997;17:1216-1223.)
Key Words: nonhuman primates • apolipoprotein E • proteoglycans • ethinyl estradiol • levonorgestrel
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
LDLs are the major transport particles of cholesterol in plasma. LDL particles are 22- to 26-nm-diameter spheres containing a surface monolayer of phospholipid, free cholesterol, and protein, and a core consisting of CE and TG.1 Plasma concentrations of LDL are strongly correlated with the incidence of CHD.2 The size of the LDL particle also has been shown to be an important contributor to development of CHD. Several studies have concluded that individuals with small, dense LDL particles are at increased risk of premature CHD.3 4 5 However, in many cases these individuals are hypertriglyceridemic, and there is an inverse relationship between LDL particle size and plasma TG concentrations in human subjects.6 More recently, it was reported that large LDL particles are more prevalent in normocholesterolemic subjects with CHD who are not overweight compared with healthy controls, and in that study LDL particle size was the best predictor of CHD prevalence.7 Several studies in nonhuman primates fed atherogenic diets have shown that LDL particle size is the best predictor of the extent and severity of coronary artery atherosclerosis.8 9 10 Therefore, factors that influence LDL particle size may affect the development of coronary artery atherosclerosis.
One factor influencing LDL particle size is contraceptive steroid treatment. Oral contraceptive users have been found to have a predominance of smaller, denser LDL subfractions compared with controls.11 Nonhuman primates treated with the oral contraceptives Ovral (EE plus norgestrel) or Demulen (EE plus LNG) had smaller LDL particles compared with untreated control animals.12 Another study in nonhuman primates demonstrated that oral contraceptive treatment resulted in a significant reduction in LDL particle size and in LDL degradation by the coronary arteries.13 In addition, several studies have shown that oral contraceptive treatment of nonhuman primates results in less coronary artery atherosclerosis.14 15 However, it is unclear from these previous studies whether the oral contraceptive treatment acts directly on LDL composition to decrease its atherogenicity. For example, we have shown in cynomolgus monkeys fed diets enriched in n-3 fatty acids that plasma LDL particles are smaller, contain fewer CE molecules, contain less apo E per particle, and bind arterial PGs less avidly compared with LDL particles from animals fed a saturated fat diet.16 17 In another study, African green monkeys fed n-3 fatty acids developed less coronary artery atherosclerosis.10 Thus, factors that reduce LDL particle size appear to influence several potential atherogenic features of LDL.
The purposes of the present study were to investigate the effect of oral contraceptive treatment of nonhuman primates on the composition and density distribution of plasma LDL and to assess the atherogenic potential of LDL using an in vitro binding assay of LDL to arterial PG. Triphasil was used as the combination oral contraceptive because it is a commonly prescribed low-dose contraceptive. Additional animals were also treated with the estrogenic component (EE) or the progestin component (LNG) alone.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals and Diet
This study involved a subset of female cynomolgus monkeys (Macaca fascicularis) from a group of monkeys used to study the effects of oral contraceptives on atherosclerosis (see below). The monkeys were imported directly from Indonesia and were quarantined for 3 months, during which time they consumed High Protein Monkey Chow. The animals were estimated to be 4 to 10 years of age and weighed 2.3 to 4.0 kg. The monkeys were fed a challenge atherogenic diet (defined as a diet that induces atherosclerosis over a period of 1 to 3 years of consumption) for 1 month to stratify monkeys into four groups with similar TPC, TG, and HDL-C responses to the atherogenic diet. The monkeys resumed eating the monkey chow diet for 6 to 8 months until they entered the experimental protocol. The atherogenic diet contained 0.28 mg cholesterol/kcal, and 44% of the total calories were from fat18 ; the diet was the same for both the challenge and experimental phases of the study. The distribution of fat calories was as follows: saturated, 46%; monounsaturated, 43%; and polyunsaturated, 11%.
A subset of 32 animals was selected for the present study. Eight animals from each treatment group were selected to represent as closely as possible the mean TPC and HDL-C concentrations (mean±SD) of the entire treatment group (n=20 to 24 per group). The subset of animals was selected after 1.5 years into the experimental phase of the study, just before blood collection for the LDL characterization analyses.
Experimental Design
Monkeys were fed either the atherogenic diet alone (control
group, n=8), the atherogenic diet plus a TOC (n=8) (Triphasil), the
estrogen component (EE) of the TOC alone (n=8), or the progestin
component (LNG) of the TOC alone (n=8). Doses were scaled for the
monkeys from the prescribed human dose based on caloric intake to
correct for differences in basal metabolic rate between the
two (ie, 1800 cal for human beings and 300 cal/d for
monkeys).14 The TOC estrogen dose was equivalent to a
human dose that varied from 30 µg EE (days 1 to 6 of the 28-day
cycle) to 40 µg (days 7 to 11), 30 µg (days 12 to 21), and no drug
(days 22 to 28). The LNG dose varied from 50 µg (days 1 to 6), 75
µg (days 7 to 11), 125 µg (days 12 to 21), and no drug (days 22 to
28). The EE and LNG groups received the same doses of the estrogen or
progestin component alone, respectively, as described for the TOC
group.
All monkeys were studied after consuming the atherogenic diet for 1.5 years. Most samples were obtained on day 21 of the 28-day treatment cycle, but nine were studied during phase 1 (days 5 to 7), and one was studied during phase 2 (day 11). Menstrual cycles were not determined for control monkeys.
Procedures involving animals were conducted in compliance with state and federal laws, standards of the US Department of Health and Human Services, and guidelines established by the Institutional Animal Care and Use Committee. Blood sampling was done while the animals were sedated with ketamine hydrochloride (10 mg/kg IM).
Lipid Analyses
Analyses of TPC, TG, and HDL-C were performed as
described previously19 ; the assays were in full
standardization with the Centers for Disease Control and
Prevention–National Heart, Lung, and Blood Institute Lipid
Standardization Program.
Lipoprotein Isolation and Density Subfractionation of LDL
Blood samples for detailed lipoprotein analyses were
taken from the animals after an overnight (18-hour) fast. Following the
administration of ketamine hydrochloride (10 mg/kg), 25
mL of blood was drawn from the femoral vein into chilled tubes (4°C)
containing 0.1% EDTA and 0.02% NaN3 (final
concentrations) at pH 7.4. LDL was isolated from plasma by
ultracentrifugation and high-performance liquid
chromatography using Superose 6B
columns.20 Cholesterol distribution of the
isolated lipoproteins was performed using enzymatic
methods.21 Isolated LDL was used for PG-binding studies
(see below) or subfractionated further by density-gradient
centrifugation.17 For LDL
subfractionation, discontinuous salt gradients were set up using 39-mL
quick-seal centrifuge tubes by first adding 10 mL of a
d=1.006 g/mL solution and then successively
underlayering 19 mL of a d=1.030 g/mL solution
(including the LDL sample) and 10 mL of a d=1.060 g/mL
solution. LDL was subfractionated using a VTi-50 vertical rotor at
242 000g at 15°C for 6 hours. Tubes were pooled to give
three density fractions of LDL: d=1.015 to 1.025
g/mL, d=1.025 to 1.035 g/mL, and
d=1.035 to 1.045 g/mL. These subfractions were
dialyzed against 0.9% NaCl, 0.01% EDTA, and 0.01% NaN3,
pH 7.4, and concentrated to 2 mL for chemical analyses. Protein
concentrations were measured using the Lowry procedure.22
Total and free cholesterol concentrations were determined
enzymatically using the Boehringer Mannheim
Diagnostic High Performance Cholesterol
Reagent (No. 236691).21 LDL esterified
cholesterol was computed by subtracting the free
cholesterol value from the total value;
cholesterol ester was obtained by multiplying the
esterified cholesterol value by 1.7. Phospholipids were
measured by the method of Fiske and SubbaRow.23 The TG
procedure was based on the enzymatic method of Fossati and
Principe.24 Plasma and subfraction apo E and B-100 (apo B)
concentrations were assayed using an enzyme-linked immunosorbent
assay.25 26 All LDL samples were stored under argon at
4°C.
PG-Binding Assay
The PG preparation used for these studies was previously
described.16 It was derived from the thoracic aortas of
two adult Macaca fascicularis monkeys by extraction with 4
mol/L guanidine HCl. Column chromatography and
density-gradient ultracentrifugation were used to
prepare a purified chondroitin sulfate PG preparation for the studies.
LDLs isolated from the plasma of cynomolgus monkeys in the four
treatment groups were used in an in vitro PG-binding assay as described
previously.16 Briefly, incubations of LDL (100 µg
cholesterol) and arterial chondroitin sulfate
PG (1 µg hexuronic acid) were performed in 1.1 mL of buffer
containing 5 mmol/L Tris, 6 mmol/L KCl, 15 mmol/L CaCl2,
and 1.5 mmol/L MgSO4 (pH 7.2) for 30 minutes at 26°C. The
resulting LDL–PG complexes were precipitated by low-speed
centrifugation (1500g for 30 minutes), the
supernatant was removed, and the pellet was resuspended in 10 µL of
1.5 mol/L NaCl and diluted to 100 µL with deionized water.
Cholesterol in the resuspended pellet was measured by an
enzymatic cholesterol assay and represented a
percentage of total LDL-C in the incubation. Previous reports have
described the specificity of the interaction, the importance of intact
PG structure, and the involvement of divalent cations in the formation
of particulate PG–LDL complexes.16 27
Data Analysis
Data are presented as the mean±SEM. Statistical
analyses were performed using the Statview SE+ program and a
Macintosh computer. One-way ANOVA was used to detect differences among
treatment groups; Fisher's post hoc least significant difference test
was used to locate the specific group differences.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The TPC, HDL-C, plasma TG, and apo B and E concentrations determined 2 years into the experimental phase of the study are shown in Table 1
. TPC concentrations were
comparable among treatment groups and ranged from 400 to 436
mg/dL. HDL-C concentrations were slightly lower for the three
treatment groups compared with the control group (33 to 37 versus 44
mg/dL), but this difference did not reach statistical
significance. The plasma TG concentrations were twofold higher in the
EE and EE+LNG groups compared with the control and LNG groups, but
because of wide interanimal variability, the differences were not
statistically significant. Plasma apo B and E concentrations were not
statistically different among the groups; however, there was a trend
toward higher apo B concentrations and lower apo E concentrations in
the EE and EE+LNG groups compared with the control and LNG groups.
|
Plasma lipoprotein cholesterol distribution values before
(pretreatment) and after 1.5 years of oral contraceptive treatment
(posttreatment) are shown in Table 2. All
lipoprotein cholesterol values were similar among the
groups of animals in the experimental subset during the pretreatment
phase of the study. After 1.5 years of oral contraceptive treatment,
there was no statistically significant difference in TPC among the four
groups. The TPC concentrations differ from those in Table 1
because the
lipoprotein cholesterol distribution was performed on a
separate blood sample taken 6 months before the one used for the
analyses in Table 1. The distribution of
cholesterol among VLDL+IDL, LDL, and HDL was significantly
affected by the experimental treatments. VLDL+IDL
cholesterol concentrations were significantly greater for
the LNG group compared with the EE and EE+LNG groups. LDL-C was
significantly higher in the EE group compared with the control and LNG
groups. However, this was not the case for the parent EE group
(LDL-C=385±34 mg/dL, n=24) compared with the parent control
group (LDL-C=367±23 mg/dL, n=24), suggesting that there may
have been a selection bias for the EE study subset with regard to LDL-C
concentrations. HDL-C concentrations were significantly lower in the EE
and LNG groups compared with the control group.
|
The effects of oral contraceptive treatment on LDL particle size and
composition were also investigated, and the results are shown in Table 3. LDL particle size (measured as LDL
molecular weight) was significantly smaller for the EE and EE+LNG
groups compared with the control and LNG groups. The number of
phospholipid and TG molecules per LDL particle was not significantly
different among diet groups, although there was a trend toward fewer
phospholipid and more TG molecules for the EE and EE+LNG groups. The
LNG group had significantly more free cholesterol molecules
per LDL particle compared with the other groups. The number of CE
molecules per LDL particle was higher for the control and LNG groups
compared with the EE and EE+LNG groups (P=.08 by post hoc
statistical analysis).
|
Having seen effects of oral contraceptive treatment on LDL
concentration and composition, we next analyzed the effects of
treatment on the density distribution of LDL subfractions. LDL
particles were subfractionated into three density fractions and
analyzed for apo B and cholesterol content. Because
each LDL particle has only one apo B molecule, the apo B distribution
was used to monitor LDL particle distribution. Most of the LDL apo B
and cholesterol were distributed in the two highest
subfractions regardless of treatment (Fig 1). However, there was significantly less
apo B and cholesterol in the d=1.015 to 1.025 g/mL
subfraction for the EE and EE+LNG groups compared with the control and
LNG groups. There was also a significant increase in apo B and
cholesterol in the d=1.025 to 1.035 g/mL subfraction
for the EE and EE+LNG groups. The distribution of apo B and
cholesterol was comparable among treatment groups for the
d=1.035 to 1.045 g/mL subfraction.
|
The percentage chemical composition of the LDL subfractions and the
unfractionated LDL (ie, total LDL) is shown in Table 4. The composition of the d=1.015 to
1.025 g/mL subfraction was most affected by the oral
contraceptive treatment. The percentage of TG was higher and the
percentage of CE lower in the EE and EE+LNG groups compared with the
control and LNG groups. In the d=1.025 to 1.035 g/mL
subfraction, the percentage of CE was lower for the EE and EE+LNG
groups. There were no significant differences in the composition of the
d=1.035 to 1.045 g/mL subfraction among treatment groups. Note
that, as expected, the percentage of CE decreased and the percentage of
protein increased as the LDL subfraction increased in density. The
composition differences among treatment groups for the total LDL
reflected those observed for the d=1.015 to 1.025 g/mL
subfraction. The LDLs from the EE and EE+LNG groups were, on average,
enriched in protein and TG and were relatively depleted of CE and free
cholesterol.
|
LDL from cynomolgus monkeys contains a significant amount of apo E, and
we previously showed that hormone replacement therapy affects the
amount of LDL apo E.28 The LDL apo E/B molar ratios for
the treatment groups of this study are shown in Table 5. Because there is a single apo B
molecule per LDL particle, the apo E/B molar ratio is a measure of the
average number of apo E molecules per LDL particle. The apo E/B molar
ratio was significantly lower for the EE and EE+LNG groups compared
with the control and LNG groups. This trend was observed for all three
LDL subfractions, although the greatest differences were seen in the
two lighter LDL subfractions. In the LNG groups, the apo E content of
the d=1.025 to 1.035 g/mL subfraction was significantly greater
than in the other three treatment groups. A similar trend was observed
for the d=1.015 to 1.025 g/mL subfraction, but the difference
between the LNG and control groups did not reach statistical
significance.
|
Because we previously observed that the amount of LDL apo E affected
its binding to arterial PGs, we performed an in vitro
binding assay using isolated LDL from each treatment group and
cynomolgus monkey arterial chondroitin sulfate PGs. The
results are shown in Fig 2. There was
nearly twice as much binding of LDL to PGs in the LNG group compared
with the other three groups. For all groups, there was a significant
correlation between the apo E/B molar ratio in the d=1.025 to 1.035
g/mL subfraction and LDL–PG binding (r=.43, n=32,
P<.01). The association between apo E/B molar ratio and
LDL–PG binding did not reach statistical significance for
unfractionated LDL or the other two subfractions of LDL.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The purpose of this study was to determine the effects of an oral contraceptive estrogen and progestin, alone or in combination, on the composition, distribution, and atherogenic potential of LDL. We found that in spite of minor changes in TPC and apo B concentrations with oral contraceptive treatment, there were significant changes in LDL. Animals treated with EE had smaller, more dense LDL particles containing fewer molecules of CE, free cholesterol, and apo E. An assay of LDL atherogenic potential also showed that LDL particles from animals treated with EE interacted less with arterial PGs than LDL particles from animals treated with LNG. In all cases the EE treatment in combination with LNG gave results similar to those of the EE alone group. Thus, oral contraceptive treatment, in particular EE, had a significant beneficial effect on the composition of LDL, resulting in a less atherogenic particle. These compositional changes in LDL may explain, in part, the decreased atherosclerosis previously observed in nonhuman primates treated with oral contraceptive steroids.14 15
Results from this and previous studies suggest that the reduction in
LDL particle size that occurs with oral contraceptive treatment is
related to the estrogen/progestin balance, with smaller LDL particles
resulting when a potent estrogen such as EE is
administered.12 13 14 Cynomolgus monkeys treated with
Demulen, which resulted in a human equivalent dose of 50 µg of EE and
1000 µg of ethynodiol diacetate per day, had smaller LDL particles
than animals treated with Ovral, which delivered 50 µg of EE and 500
µg of norgestrel per day.12 14 Thus, even though
the dosage of EE was the same, the less potent progestin in the Demulen
treatment group resulted in a higher estrogen/progestin balance and a
lower average LDL molecular weight compared with the Ovral treatment
group. In another study in which Ovral (50 µg EE+500 µg
norgestrel) was compared with TOC treatment (30 to 40 µg
EE+50 to 125 µg LNG), the TOC group had smaller LDL
particles.13 This likely resulted because the dosage of
active progestin in the Ovral group (equivalent to 250 µg LNG) was
greater than in the TOC group (50 to 125 µg LNG), whereas the dosage
of EE was similar between the two groups (50 versus 30 to 40 µg EE,
respectively). In the present study, EE treatment alone resulted in
significantly smaller LDL particles, whereas LNG alone resulted in a
higher average LDL particle size (Table 3). Similar to our previous
study,13 the combination TOC treatment also resulted in
significantly smaller LDL particles. The results taken together
demonstrate that the estrogen/progestin balance is inversely related to
LDL particle size in cynomolgus monkeys.
Results from several studies suggest a mechanistic link between EE
treatment and smaller plasma LDL particles. A strong positive
correlation between hepatic CE content and plasma LDL size has been
observed in nonhuman primates,19 and hepatic CE content
was reduced in animals given an oral contraceptive containing EE and
norgestrel or LNG compared with untreated
controls.29 Oral contraceptive treatment has been shown to
stimulate biliary secretion of cholesterol in
women30 and to increase hepatic 7-
hydroxylase, the
rate-limiting enzyme for bile acid synthesis, in baboons compared with
progesterone treatment alone or no treatment.31 The
results of these studies suggest that estrogen treatment may route
hepatic cholesterol into the bile acid biosynthetic
pathway, decreasing the available hepatic cholesterol pool
for CE storage and for secretion into VLDL. This, in turn, would result
in CE-depleted VLDL being converted into LDL particles in plasma that
subsequently would be relatively poor in CE and of smaller size than in
untreated animals.
In nonhuman primates large LDL particles are associated with increased coronary artery atherosclerosis,8 32 33 whereas small LDL particles are associated with increased CHD in human subjects.34 35 One potential explanation for this difference is related to the low concentrations of plasma TGs in nonhuman primates (20 to 30 mg/dL), which is likely due to their relatively high lipoprotein lipase activity.36 In humans there is adequate exchange of VLDL TG for LDL CE by CE transfer protein to enrich LDL with TG. Subsequent hydrolysis of the LDL TG by hepatic or lipoprotein lipase results in smaller LDL particles.37 This process cannot occur efficiently in nonhuman primates because of low plasma TG concentrations, so LDL particles become enriched in CE and larger compared with LDL particles from human beings. Despite this difference, both nonhuman and human primates respond to estrogen therapy with a reduction in LDL particle size.38 39 40 Because estrogen replacement therapy reduces the risk of CHD in women, even though LDL particle size is apparently reduced,41 42 small LDL particles in women, per se, do not appear to increase the risk of CHD. Alternatively, estrogen therapy may minimize the atherogenic effect of small LDL particles by some other means, such as altering the interaction of LDL at the vessel wall.
Arterial degradation of LDL has been positively correlated
with LDL particle size in cynomolgus monkeys, and monkeys receiving
combination oral contraceptive treatment had smaller LDL particles and
less arterial LDL degradation compared with untreated
controls.13 One potential explanation for this outcome
could be decreased binding of LDL from animals treated with
contraceptive hormones to arterial PGs, a
glycosaminoglycan-enriched macromolecular complex
thought to be involved in trapping plasma LDL in the artery
wall.43 44 45 Incubation of PG–LDL complexes with cells in
culture results in massive intracellular lipid accumulation that may
lead to foam cell formation in vivo.46 47 Previously, we
showed that large, apo E-enriched LDL particles preferentially bind to
arterial chondroitin sulfate PGs in vitro compared with
small, apo E-poor LDL particles, and part of the increased binding was
related to the apo E content of the LDL.16 17 In addition,
we have shown that apo E is responsible for an increased binding
affinity of large LDL particles from cynomolgus monkeys to fibroblasts
in culture.48 In the present study we found a twofold
increase in binding of LDL from the LNG group to PG compared with the
other groups. The difference in LDL binding to PG seemed unrelated to
the apo E content of total LDL because the control and LNG groups had
similar values (Table 5). However, the amount of apo E in the d=1.025
to 1.035 g/mL subfraction was significantly higher in the LNG
group compared with the other groups, and this is the subfraction of
LDL that contains the majority of the cholesterol and apo B
(Fig 1). There was also a statistically significant correlation
(r=.43, P<.01) between the apo E/B molar ratio
in the d=1.025 to 1.035 g/mL subfraction and the percentage of
LDL cholesterol bound to PG. In a previous study, apo E
content of an LDL subfraction (d=1.015 to 1.025 g/mL) was highly
correlated with the ability to bind to arterial
PG.17 Together these results suggest that a likely
explanation for the increased binding of LDL to arterial PG
in the LNG group is related to the increased apo E content.
The animals in this study responded to EE treatment alone or in
combination with LNG with an increase in plasma and LDL TG and a
decrease in apo E. The changes in LDL were particularly apparent in the
d=1.015 to 1.025 g/mL subfraction (Tables 4 and 5). Estrogen
treatment is known to inhibit TG hydrolysis by hepatic
lipase.49 50 The selective increase of TG in the d=1.015
to 1.025 g/mL subfraction may result if hepatic lipase
preferentially hydrolyzes TG in the largest, least dense LDL
subfractions. The d=1.015 to 1.025 g/mL LDL particle is enriched
in apo E compared with the other two subfractions (Table 5), and
hepatic lipase activity is stimulated by apo E.51 We
hypothesize that the preferential increase of TG in the d=1.015 to
1.025 g/mL subfraction of LDL in EE-treated cynomolgus monkeys
may result from the inhibition of hepatic lipase by estrogen as well as
reduced activation of hepatic lipase resulting from a decrease in the
average amount of apo E per LDL particle. We observed similar results
in another study of surgically postmenopausal cynomolgus monkeys given
conjugated equine estrogens.28 The decreased LDL apo E
seen in EE-treated animals may have resulted as a general effect of
estrogen on plasma apo E concentrations, as has been described for
women50 and baboons.52 Total plasma apo E
concentrations were 33% to 36% lower for the EE and EE+LNG groups
(Table 1), and LDL apo E values averaged 60% to 67% lower compared
with the control group. These data suggest that treatment of monkeys
with EE alone or in combination with LNG may reduce the amount of
available apo E binding to the LDL particle surface. Alternatively, the
decrease in LDL size in estrogen-treated animals may be caused by
decreased hepatic secretion of CE, resulting in smaller plasma
LDL.19 These smaller LDL particles may be relatively
poorer substrates for hepatic lipase than larger LDL, resulting in TG
enrichment of the smaller LDL particles in the EE-treated group.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported, in part, by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL-45666, HL-08373, HL-49373, and HL-25161), a National Research Service Award (J.M.M.),and a grant from the Center for Research, School of Science and Health, William Paterson College of New Jersey, Wayne, NJ (J.M.M.). We gratefully acknowledge Linda Odham for her assistance in manuscript preparation and Karen Klein for editorial comments.
Received April 23, 1996; accepted October 2, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Deckelbaum RJ, Shipley GG, Small DM. Structure and interaction of lipids in human plasma low density lipoproteins. J Biol Chem. 1977;252:744-754.
2.
The Lipid Research Clinics Coronary Primary
Prevention Trial results. II. The relationship of reduction in
incidence of coronary heart disease to cholesterol
lowering. JAMA. 1984;251:365-374.
3. Austin MA. Small, dense low-density lipoprotein as a risk factor for coronary heart disease. Int J Clin Lab Res. 1994;24:187-192.[Medline] [Order article via Infotrieve]
4. O'Brien R. Biological importance of low-density-lipoprotein subfractions. J Cardiovasc Risk. 1994;1:207-211.[Medline] [Order article via Infotrieve]
5. Krauss RM. Dense low density lipoproteins and coronary artery disease. Am J Cardiol. 1995;75:53B-57B.[Medline] [Order article via Infotrieve]
6. Crouse JR, Parks JS, Schey HM, Kahl FR. Studies of low density lipoprotein molecular weight in human beings with coronary artery disease. J Lipid Res. 1985;26:566-574.[Abstract]
7. Tallis GA, Shephard MDS, Sobecki S, Whiting MJ. The total apolipoprotein B/LDL-cholesterol ratio does not predict LDL particle size. Clin Chim Acta. 1995;240:63-73.[Medline] [Order article via Infotrieve]
8. Rudel LL, Bond MG, Bullock BC. LDL heterogeneity and atherosclerosis in nonhuman primates. Ann N Y Acad Sci U S A. 1985;454:248-253.[Medline] [Order article via Infotrieve]
9.
Rudel LL, Leathers CW, Bond MG, Bullock BC.
Dietary ethanol-induced modifications in
hyperlipoproteinemia and
atherosclerosis in nonhuman primates (Macaca
nemestrina).
Arteriosclerosis. 1981;1:144-155.
10.
Parks JS, Kaduck-Sawyer J, Bullock BC, Rudel LL.
Effect of dietary fish oil on coronary artery and aortic
atherosclerosis in African green monkeys.
Arteriosclerosis. 1990;10:1102-1112.
11. de Graaf J, Swinkels DW, Demacker PN, de Haan AF, Stalenhoef AF. Differences in the low density lipoprotein subfraction profile between oral contraceptive users and controls. J Clin Endocrinol Metab. 1993;76:197-202.[Abstract]
12.
Parks JS, Pelkey SJ, Babiak J, Clarkson TB.
Contraceptive steroid effects on lipids and lipoproteins in cynomolgus
monkeys. Arteriosclerosis. 1989;9:261-268.
13.
Wagner JD, Adams MR, Schwenke DC, Clarkson TB.
Oral contraceptive treatment decreases arterial low density
lipoprotein degradation in female cynomolgus monkeys.
Circ Res. 1993;72:1300-1307.
14. Adams MR, Clarkson TB, Shively CA, Parks JS, Kaplan JR. Oral contraceptives, lipoproteins, and atherosclerosis. Am J Obstet Gynecol. 1990;163:1388-1393.[Medline] [Order article via Infotrieve]
15. Clarkson TB, Shively CA, Morgan TM, Koritnik DR, Adams MR, Kaplan JR. Oral contraceptives and coronary artery atherosclerosis of cynomolgus monkeys. Obstet Gynecol. 1990;75:217-222.[Medline] [Order article via Infotrieve]
16.
Edwards IJ, Gebre AK, Wagner WD, Parks JS.
Reduced proteoglycan binding of low density lipoproteins from monkeys
(Macaca fascicularis) fed a fish oil versus lard
diet. Arterioscler Thromb. 1991;11:1778-1785.
17. Parks JS, Gebre AK, Edwards IJ, Wagner WD. Role of LDL subfraction heterogeneity in the reduced binding of low density lipoproteins to arterial proteoglycans in cynomolgus monkeys fed a fish oil diet. J Lipid Res. 1991;32:2001-2008.[Abstract]
18.
Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS,
Wolfe MS, Clarkson TB. Inhibition of coronary artery
atherosclerosis by 17-beta estradiol in ovariectomized
monkeys: lack of an effect of added progesterone.
Arteriosclerosis. 1990;10:1051-1057.
19. Parks JS, Wilson MD, Johnson FL, Rudel LL. Fish oil decreases hepatic cholesteryl ester secretion but not apo B secretion in African green monkeys. J Lipid Res. 1989;30:1535-1544.[Abstract]
20. Parks JS, Gebre AK. Studies on the effect of dietary fish oil on the physical and chemical properties of low density lipoproteins in cynomolgus monkeys. J Lipid Res. 1991;32:305-315.[Abstract]
21. Auerbach BJ, Parks JS, Applebaum-Bowden D. A rapid and sensitive micro-assay for the enzymatic determination of plasma and lipoprotein cholesterol. J Lipid Res. 1990;31:738-742.[Abstract]
22.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin phenol reagent. J
Biol Chem. 1951;193:265-275.
23. Fiske CH, SubbaRow Y. Colorimetric determination of phosphorus. J Biol Chem. 1925;66:357-400.
24.
Fossati P, Principe L. Serum
triglycerides determined colorimetrically
with an enzyme that produces hydrogen peroxide. Clin
Chem. 1982;28:2077-2080.
25. Koritnik DL, Rudel LL. Measurement of apolipoprotein A-I concentration in nonhuman primate serum by enzyme-linked immunosorbent assay (ELISA). J Lipid Res. 1983;24:1639-1645.[Abstract]
26.
Sorci-Thomas M, Wilson MD, Johnson FL, Williams DL,
Rudel LL. Studies on the expression of genes encoding
apolipoproteins B100 and B48 and the low density lipoprotein receptor
in nonhuman primates: comparison of dietary fat and
cholesterol. J Biol Chem. 1989;264:9039-9045.
27. Steele RH, Wagner WD, Rowe HA, Edwards IJ. Artery wall derived proteoglycan-plasma lipoprotein interaction: lipoprotein binding properties of extracted proteoglycans. Atherosclerosis. 1987;65:51-62.[Medline] [Order article via Infotrieve]
28. Manning JM, Campos G, Edwards IJ, Wagner WD, Wagner JD, Adams MR, Parks JS. Effects of hormone replacement modalities on low density lipoprotein composition and distribution in ovariectomized cynomolgus monkeys. Atherosclerosis. 1996;121:217-229.[Medline] [Order article via Infotrieve]
29.
Colvin PL Jr, Wagner JD, Heuser MD, Sorci-Thomas
MG. Oral contraceptives decrease hepatic cholesterol
independent of the LDL receptor in nonhuman primates.
Arterioscler Thromb. 1993;13:1645-1649.
30. Everson GT, Fennessey P, Kern F Jr. Contraceptive steroids alter the steady-state kinetics of bile acids. J Lipid Res. 1988;29:68-76.[Abstract]
31. Kushwaha RS, Born KM. Effect of estrogen and progesterone on the hepatic cholesterol 7-alpha-hydroxylase activity in ovariectomized baboons. Biochim Biophys Acta. 1991;1084:300-302.[Medline] [Order article via Infotrieve]
32. Rudel LL, Parks JS, Johnson FL, Babiak J. Low density lipoproteins in atherosclerosis. J Lipid Res. 1986;27:465-474.[Medline] [Order article via Infotrieve]
33. Rudel LL, Sawyer JK, Parks JS. Dietary fat, lipoprotein structure, and atherosclerosis in primates. Atheroscler Rev. 1991;23:41-50.
34.
Sniderman A, Shapiro S, Marpole D, Skinner B, Teng B,
Kwiterovich PO Jr. Association of coronary
atherosclerosis with hyperapo betalipoproteinemia
(increased protein but normal cholesterol levels in human
plasma low density lipoproteins). Proc Natl Acad Sci
U S A. 1980;77:604-608.
35.
Austin MA, Breslow JL, Hennekens CH, Buring JE, Willett
WC, Krauss RM. Low-density lipoprotein subclass patterns and
risk of myocardial infarction. JAMA. 1988;260:1917-1921.
36.
Rudel LL, Star RJ. Species, diet, and gender
differences in plasma postheparin lipolytic activities in
nonhuman primates: relationships with plasma lipids and high density
lipoproteins. Arteriosclerosis. 1990;10:350-357.
37.
Deckelbaum RJ, Eisenberg S, Oschry Y, Butbul E, Sharon
I, Olivecrona T. Reversible modification of human plasma low
density lipoproteins towards triglyceride-rich
precursors. J Biol Chem. 1982;257:6509-6517.
38. Granfone A, Campos H, McNamara JR, Schaefer MM, Lamon-Fava S, Ordovas JM, Schaefer EJ. Effects of estrogen replacement on plasma lipoproteins and apolipoproteins in post menopausal, dyslipidemic women. Metabolism. 1992;41:1193-1198.[Medline] [Order article via Infotrieve]
39. Campos H, Sacks FM, Walsh BW, Schiff I, O'Hanesian MA, Krauss RM. Differential effects of estrogen on low-density lipoprotein subclasses in healthy postmenopausal women. Metabolism. 1993;42:1153-1158.[Medline] [Order article via Infotrieve]
40. Wagner JD, Clarkson TB, St Clair RW, Schwenke DC, Shively CA, Adams MR. Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest. 1991;88:1995-2002.
41. LaRosa JC. Estrogen: risk versus benefit for the prevention of coronary artery disease. Coron Artery Dis. 1993;4:588-594.[Medline] [Order article via Infotrieve]
42. Seed M, Crook D. Post-menopausal hormone replacement therapy, coronary heart disease and plasma lipoproteins. Curr Opin Lipid. 1994;5:48-58.[Medline] [Order article via Infotrieve]
43. Mawhinney TP, Augustyn JM, Fritz KE. Glycosaminoglycan-lipoprotein complexes from aortas of hypercholesterolemic rabbits: Part 1. Isolation and characterization. Atherosclerosis. 1978;31:155-167.[Medline] [Order article via Infotrieve]
44. Srinivasan SR, Radhakrishnamurthy B, Pargaonkar PS, Berenson GS, Dolan P. Lipoprotein-acid mucopolysaccharide complexes of human atherosclerotic lesions. Biochim Biophys Acta. 1975;388:58-70.[Medline] [Order article via Infotrieve]
45. Walton KW, Williamson N. Histological and immunofluorescent studies on the evolution of the human atheromatous plaque. J Atheroscler Res. 1968;8:599-624.[Medline] [Order article via Infotrieve]
46. Salisbury BGJ, Falcone DJ, Minick CR. Insoluble low-density lipoprotein-proteoglycan complexes enhance cholesteryl ester accumulation in macrophages. Am J Pathol. 1985;120:6-11.[Abstract]
47.
Vijayagopal P, Srinivasan SR, Radhakrishnamurthy B,
Berenson GS. Lipoprotein-proteoglycan complexes from
atherosclerotic lesions promote cholesteryl ester accumulation in human
monocytes/macrophages. Arterioscler Thromb. 1992;12:237-249.
48. Linga V, Leight MA, Curtiss LK, Marcel YL, St Clair RW, Parks JS. Dietary fish oil-induced decrease in low density lipoprotein binding to fibroblasts is mediated by apolipoprotein E. J Lipid Res. 1994;35:491-500.[Abstract]
49. Kushwaha RS, Foster DM, Murthy VN, Carey KD, Bernard MG. Metabolic regulation of apoproteins of high-density lipoproteins by estrogen and progesterone in the baboon (Papio sp). Metabolism. 1990;39:544-552.[Medline] [Order article via Infotrieve]
50. Applebaum-Bowden D, McLean P, Steinmetz A, Fontana D, Matthys C, Warnick GR, Cheung M, Albers JJ, Hazzard WR. Lipoprotein, apolipoprotein, and lipolytic enzyme changes following estrogen administration in postmenopausal women. J Lipid Res. 1989;30:1895-1906.[Abstract]
51. Thuren T, Weisgraber KH, Sisson P, Waite M. Role of apolipoprotein E in hepatic lipase catalyzed hydrolysis of phospholipid in high-density lipoproteins. Biochemistry. 1992;31:2332-2338.[Medline] [Order article via Infotrieve]
52. Kushwaha RS, Foster DM, Barrett PH, Carey KD, Bernard MG. Metabolic regulation of plasma apolipoprotein E by estrogen and progesterone in the baboon (Papio sp). Metabolism. 1991;40:93-100.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  D.-H. Kang, E. S. Yu, K.-I. Yoon, and R. Johnson The Impact of Gender on Progression of Renal Disease: Potential Role of Estrogen-Mediated Vascular Endothelial Growth Factor Regulation and Vascular Protection Am. J. Pathol., February 1, 2004; 164(2): 679 - 688. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||