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Original Studies |
Departments of Clinical Chemistry (K.W., M.M.H., I.F., H.W., W.M.), Obstetrics and Gynecology (B.W., M.K., H.P.Z.), and Sports Medicine (M.W.B.), University of Freiburg, D-79106 Freiburg, Germany
Address all correspondence and requests for reprints to: Karl Winkler, M.D., Department of Clinical Chemistry, Albert Ludwigs University School of Medicine, Hugstetter Strasse 55, D-79106 Freiburg, Germany. E-mail: kwinkler{at}ukl.uni-freiburg.de
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Abstract |
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Introduction |
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As a consequence, the maternal lipid metabolism is specifically altered during pregnancy. Cholesterol and phospholipids increase moderately, whereas plasma triglyceride levels rise markedly (2, 3). High amounts of triglycerides are not only found in the very low density lipoprotein (VLDL) fraction, but in all lipoprotein fractions [low density lipoprotein (LDL) and high density lipoprotein (HDL)] during late gestation (4, 5). Two mechanisms specific for pregnancy seem to be responsible for this phenomenon. First, elevated estrogen levels during gestation result in an increased hepatic synthesis of triglyceride-rich VLDL (6, 7). Secondly, removal of lipoprotein triglycerides is reduced due to low activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HL), the effect being more striking for HL than for LPL (8, 9, 10). The abundance of VLDL triglycerides drives an accelerated transfer of triglycerides to lipoproteins of higher density by the cholesteryl ester transfer protein (CETP) (11, 12). Thus, the reduced HL activity appears to be responsible for the shift of HDL subclasses toward larger, triglyceride-rich, and more buoyant species in late gestation (9).
During gestation, LDL particles become enriched in triglycerides as well. However, in contrast to HDL particles, LDL particles have been reported to become smaller and denser (10, 13, 14). LDL particles are heterogeneous with regard to their chemical and physical properties (15, 16). Using nondenaturing gradient gel electrophoresis, Austin et al. (17) demonstrated that two patterns of LDL subclass distribution, A and B, can be distinguished. Pattern A is characterized by a predominance of LDL particles that are large and buoyant, and pattern B is characterized by a predominance of small, dense LDL particles (18). The larger, more buoyant subclasses of LDL predominate in healthy females of reproductive age, whereas smaller, denser LDL often occur after menopause (19). Compared with large and buoyant LDL, small dense LDL particles are more susceptible to oxidation, show increased binding to proteoglycans of the vessel wall, and exhibit reduced uptake by the LDL receptor (20). In men and nonpregnant women, plasma triglycerides account for 40–60% of the variability in small, dense LDL concentrations (21, 22, 23). Several studies have shown there to be an association between elevated plasma triglyceride concentrations, small dense LDL (pattern B) (24, 25, 26) and decreased HDL cholesterol (25, 27), in particular HDL2 cholesterol (24). This metabolic situation is referred to as atherogenic lipoprotein phenotype (24). Thus, elevated triglycerides and the accumulation of small, dense LDL during pregnancy are thought to increase the risk for endothelial damage (28, 29) despite the fact that there is a preponderance of large, buoyant HDL in late gestation (9, 13).
To address the question whether lipid metabolism during pregnancy is indeed similar to that described for the atherogenic lipoprotein phenotype, we examined LDL subfractions by equilibrium density gradient ultracentrifugation during gestation. By including 23 pregnant women, this study is the largest prospective study investigating LDL subfractions.
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Materials and Methods |
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Twenty-three pregnant women were studied three times during their visits to the prenatal clinic during the first, second, and third trimesters. The women were not sampled in a defined nutritional status, because fasting conditions are not well tolerated during pregnancy. The control group included 15 healthy, nonpregnant women with regular menstrual cycles. None of the subjects was receiving medication known to influence lipid metabolism. Blood samples were taken from the control group in the midluteal phase when, in the case of conception, implantation takes place and pregnancy begins. This study was approved by the ethics review committee of the University of Freiburg. Informed consent was obtained from each subject, and all procedures were in accordance with the Helsinki Declaration of 1975, revised in 1983.
Lipoprotein separation
Lipoproteins were isolated by sequential preparative ultracentrifugation using the following densities: density less than 1.006 kg/L for VLDL, density between 1.006–1.019 for intermediate density lipoprotein (IDL), density between 1.019–1.063 kg/L for LDL, and density between 1.063–1.21 for HDL (30). LDL subfractions were separated according to the method of Baumstark et al. (31). Total LDL (density, 1.019–1.063 kg/L) were fractionated into six density classes by equilibrium density gradient centrifugation. Density ranges of the subfractions were: LDL-1, less than 1.031 kg/L; LDL-2, 1.031–1.034 kg/L; LDL-3, 1.034–1.037 kg/L; LDL-4, 1.037–1.040 kg/L; LDL-5, 1.040–1.044 kg/L; and LDL-6, more than 1.044 kg/L. All centrifugation steps were carried out at 18 C using partially filled polycarbonate bottles (6 mL) in a 50-Ti rotor. Variability in LDL subfractions with respect to nutritional status were assessed in 5 probands sampled after an overnight fast, 3 h after breakfast, and 3 h after lunch. The average of the intraindividual coefficients of variance (CVs) of each individual for apolipoprotein B (apoB) were 9.6% and 10.2% for VLDL and IDL, and 4.4%, 5.0%, 4.2%, 2.5%, 3.6%, and 3.2% for LDL-1 through LDL-6, respectively. In addition, 6 females were sampled longitudinally in the luteal phase over an average period of 52 ± 30 days, ranging from 24–108 days. Each individual was sampled 2–4 times, and the average of the intraindividual CVs for apoB were 28.0% and 28.1% for VLDL and IDL, and 7.3%, 8.8%, 7.5%, 5.4%, 6.7%, and 8.9% for LDL-1 through LDL-6, respectively.
Lipoprotein chemistry
Cholesterol (CH), free cholesterol, triglycerides (TG), and phospholipids (PL) were determined enzymatically with the CHOD-PAP, GPO-PAP, and PLD-PAP methods (Roche Diagnostics, Mannheim, Germany), respectively. The concentration of esterified cholesterol (CE) was calculated as the difference between total cholesterol and free cholesterol. Concentrations of apolipoproteins were determined by turbidimetry on a Wako 30R analyzer (Wako Chemicals, Tokyo, Japan) using specific polyclonal antisera (Rolf Greiner Biochemica, Frickenhausen, Germany) for the respective antigen. The interassay CVs ranged between 1.0–6.6% for the lipid measurements and between 2.4–8.0% for the apoprotein measurements, respectively.
Particle radius
Lipoprotein radii were calculated using the molar concentrations of free cholesterol, cholesterol esters, phospholipids, triglycerides, and apoB-100 of the respective density fractions (31).
CETP activity
The activity of the cholesteryl ester transfer protein was measured with a fluorescence-based assay (Diagnescent Technologies, Inc., Bronxville, NY). The interassay CV was below 10.0%.
Estradiol
Estradiol levels were determined with a commercial RIA (Biochem Immunosystems GmbH, Freiburg, Germany). The interassay CV was 7.5%.
Statistical analysis
Differences between the luteal phase (control group) and the first, second, and third trimesters were tested for significance using the nonparametric, unpaired Kruskal-Wallis method of one-way ANOVA on ranks. Comparisons between groups were performed using Dunn’s test. Comparisons of each of the three trimesters vs. the control group were made using Dunn’s method vs. the control as indicated. Differences between groups were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). All women had uncomplicated
pregnancies and gave birth, on the average, at week 39.5 ± 1.4.
The average birth weight of the babies was 3569 ± 484 g.
Both control and pregnant groups included only Caucasian women. There
was no difference between the control group and the pregnant group
before the onset of pregnancy with regard to age and body mass index
(Table 1).
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).
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). Similarly, the
concentrations of IDL-CH, IDL-TG, and IDL-apoB rose significantly, with
the greatest increase observed from the second to the third trimester
(Table 4). LDL-CH, LDL-TG, and LDL-apoB
were significantly higher in the first trimester compared with the
nonpregnant state and showed a further increase as gestation advanced
(Table 5).
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. Each apoB-100-containing
lipoprotein possesses only one apoB-100 molecule. The concentration of
apoB-100 in each density fraction, therefore, represents the number of
lipoprotein particles present in this particular fraction. Compared
with the nonpregnant state, the concentration of LDL-2 was low in the
first trimester, whereas the concentration of LDL-6 was significantly
higher. The concentration of the LDL-6 fraction maintained virtually
the same level in the second and third trimesters. In the second
trimester the concentrations of VLDL, IDL, and LDL-1 through LDL-5
further increased, producing a distribution of lipoproteins dominated
by buoyant lipoprotein species, namely VLDL, IDL, and in particular
LDL-1. Toward delivery VLDL, IDL, and LDL-1 increased even further,
with the concentration of LDL-1 clearly dominating. LDL particles
became significantly enriched in triglycerides, as indicated by the
molar ratio of triglycerides to apoB in LDL (Table 5
). This was also
observed for each of the LDL subfractions (Fig. 2). The increase in LDL-TG was
accompanied by significant decreases in the cholesterol and
phospholipid content per LDL particle thus resulting in a decrease of
the average particle size of LDL (Table 5). The decrease in particle
size was also observed in each of the LDL subfractions, the
difference of LDL-3 being statistically significant compared with the
nonpregnant control group (Fig. 3).
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).
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shows the ratio of CE to TG in
VLDL (A), IDL (B), LDL (C), and HDL (D) plotted vs. the time
of gestation. Regression analysis revealed that the VLDL-CE/TG-ratio
correlated significantly (r = 0.223; P = 0.047)
with the time of gestation. The IDL-CE/TG ratio did not correlate
(r = 0.073); however, there was a tendency toward increased values
with advancing gestation. In striking contrast, the CE/TG ratios of LDL
(r = -0.726; P < 0.0001) and HDL (r =
-0.713; P < 0.0001) were inversely correlated with
the time of gestation.
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). Compared with the luteal phase, the
concentration of free fatty acids was higher in the first trimester and
increased continuously toward the end of pregnancy, although none of
these changes was significant (Table 7). As previously described
(9), the activity of CETP increased during pregnancy, but
none of the changes was significant (Table 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood samples were not drawn under defined nutritional conditions. However, the impact of nutrition on the variability of LDL subfractions was on the order of the interassay variability in apoB and may, therefore, be neglected. This was an expected finding, as LDL have a residence time of several days. Thus, short-term disturbances of apoB metabolism such as nutrition are not likely to influence LDL subfraction distribution. Compared to nonpregnant women, LDL-6, the densest LDL subfraction, was significantly higher in the first trimester and remained at this same level throughout pregnancy. However, with advancing gestation, the LDL distribution pattern became increasingly dominated by triglyceride-rich lipoproteins, namely VLDL, IDL, and in particular the most buoyant LDL (LDL-1) in the 2nd and 3rd trimesters. This was an unexpected finding, because the few previous studies reported the preponderance of small, dense LDL throughout all stages of pregnancy, including late gestation (10, 13, 14). Using nondenaturing PAGE, Silliman et al. (13) studied 36 Hispanic women in the 36th week of gestation. Among these, 1 individual showed pattern A (mainly large LDL), 18 showed the intermediate pattern I, and 17 showed pattern B (mainly small LDL). Using the same method, Hubel et al. (14) investigated the LDL peak particle diameter on 4 occasions during pregnancy and twice postpartum. In 7 of 10 women a progressive decrease in LDL particle size was seen during normal gestation as TG increased, followed by the reversal of these changes by 6–12 weeks postpartum. Sattar et al. (10) used nonequilibrium density gradient ultracentrifugation to separate LDL by flotation rate into 3 subfractions (LDL-I, LDL-II, and LDL-III) (35) at 5-week intervals between the 10th and 35th week of gestation. In 6 of 10 women an increase in LDL-III mass at the expense of LDL-II was reported, with the proportion of LDL-I remaining unchanged. However, in the other four subjects no change in the LDL subfraction pattern occurred throughout gestation.
The overt discrepancy between the previously published data and the observations reported here may be due to the different methodology used. Nondenaturing gradient gel electrophoresis separates LDL by virtue of size and provides an estimate of the particle diameter of the major LDL peak (17). The density gradient ultracentrifugation used by Sattar et al. does not reach isopycnic equilibrium (35) and therefore separates LDL by virtue of flotation rate. Flotation rate depends on both particle buoyancy and the size of the particle. It thus appears that all studies conducted previously examined the LDL subfraction profile with methods depending more or less on particle size. In contrast, the method used in this study relies upon equilibrium density gradient ultracentrifugation, which separates LDL subfractions by virtue of their density (31).
The increase in TG per LDL particle with the decrease in cholesterol and phospholipid content at the same time suggests that a change in the size of a lipoprotein particle may not necessarily be accompanied by a matching change in density. To address this possibility, we calculated the radii of the LDL particles from the lipid and apolipoprotein compositions measured in each of the separated LDL subfractions (31). The particle size of all LDL subfractions continuously decreased between the luteal phase and the third trimester; the change in the LDL-3 fraction was significant compared with that during the luteal phase. As LDL-3 particles are medium in density and in size, the changes in size seen in this fraction may correspond to the changes in the peak LDL particle diameters reported by Silliman and Hubel (13, 14), in agreement with our findings.
What is the mechanism underlying the changes of lipoprotein metabolism observed during pregnancy? As previously discussed (10, 13, 14), the elevated TG levels already present in the first trimester may be responsible for the increase in dense LDL seen during the early stages of pregnancy. However, with advancing gestation there is no further increase in dense LDL. In contrast, there is an increase in buoyant lipoproteins, namely VLDL, IDL, and LDL-1, a pattern closely resembling the situation in HL deficiency. HL has been reported to be involved in the conversion of IDL to LDL (36, 37), in chylomicron remnant catabolism (38, 39), and in HDL metabolism (40, 41). Impaired HL activity results in elevated HDL2-CH (42, 43, 44) and an enrichment of LDL and HDL particles with triglycerides (43, 44, 45).
Alvarez et al. (9) investigated the HDL metabolism of 25 pregnant women during gestation and postpartum. The TG/CH ratio in both LDL and HDL was shown to increase due to the activity of CETP, although the CETP activity did not increase significantly with advancing gestation. Similar observations were made in the present study. Although not analyzed here, the enrichment of HDL particles with TG is likely to represent an increase in the more buoyant HDL2 particles. This is in line with studies that showed that the increase in HDL during late pregnancy is associated with a dramatic increase in the most buoyant HDL species, HDL2b, and a significant decrease in HDL3 levels (11). In the study by Alvarez et al., the postheparin HL activity progressively decreased after the first trimester throughout pregnancy and was significantly correlated with the changes in the HDL subclasses (9). In addition, HL activity was negatively correlated with estradiol levels (9), which is in line with other studies indicating that elevated estrogen concentrations are associated with decreased HL activities (8, 10, 46). The significant increase in apoC-III, an inhibitor of LPL, may also contribute to the impairment of maternal lipolysis. As a consequence this may delay the turnover and increase the residence time of TG-rich lipoproteins. The prolonged exposure of these lipoproteins to CETP rather than elevated activities of CETP might, therefore, be responsible for the observed compositional changes: VLDL and IDL particles become enriched in CE, whereas LDL and HDL particles become enriched in triglycerides. Thus, in late pregnancy, the observed changes in lipoprotein composition may reflect a reduced catabolism of TG-rich lipoproteins. In contrast, treatment with low doses of estrogen as in hormone replacement therapy (47) or hormonal contraception (48) results in an increased production of large, TG-rich VLDL only, whereas VLDL catabolism is not affected.
What may be the implications of the metabolic changes reported here for
the physiology of pregnancy? Sattar et al. (49)
found that in mothers with intrauterine growth retardation the
appropriate synthesis of LDL precursors, namely VLDL and IDL, fails to
occur in the third trimester. This suggests that in late gestation the
welfare of the unborn child depends on an adequate supply of lipids. In
the fetus, longer chain essential fatty acids are synthesized by the
liver and the brain from linoleic acid and 
-linolenic acid, which
are provided by the mother (50). Free fatty acids are
transferred across the placenta by simple diffusion (51, 52). However, the capacity of this transport is limited.
Therefore, the increasing requirements of the fetus in late gestation
need to be satisfied by additional means. Human placenta is known to
express lipoprotein receptors in high amounts (53, 54).
Interestingly, the binding of VLDL to placental membranes exceeds that
of LDL, suggesting that the placenta is primarily endowed with
receptors that preferentially bind to VLDL (55). In this
regard, the VLDL/apoE receptor appears to be of particular interest.
The localization and regulation of this receptor suggest a major role
in placental lipid transport. Northern blot analysis of placenta
revealed a 2.6-fold increase in VLDL/apoE receptor messenger
ribonucleic acid between the first trimester and delivery
(54). Thus, in the catabolic phase of pregnancy, the
effect of placental hormones is to enhance VLDL production and decrease
HL activity. Together with the increased expression of the VLDL/apoE
receptor in the placenta, this may result in a coordinated rerouting of
the swelling flux of TG-rich lipoproteins from the mother to the
feto-placental unit (Fig. 5).
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Acknowledgments |
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Received December 30, 1999.
Revised June 6, 2000.
Revised August 15, 2000.
Accepted August 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phe and
Thr383Met: correlation between hepatic lipase
activity and phenotypic expression. J Lipid Res. 37:825–834.[Abstract]
2-macroglobulin receptor. FEBS
Lett. 255:275–280.[CrossRef][Medline]
G mutation in intron I of the
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deficiency. J Lipid Res. 37:1213–1223.[Abstract]
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, Luteal phase; •, first trimester;
, second trimester; 
, third trimester. By ANOVA: a,
P < 0.05 vs. luteal phase; b,
P < 0.05 vs. first trimester; c,
P < 0.05 vs. second trimester.
