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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, Kochi Medical School, Kochi, Japan.
Address reprint requests to: Akihiko Wakatsuki, MD, Kochi Medical School, Department of Obstetrics and Gynecology, Oko-cho, Nankoku, Kochi, 783-8505, Japan
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Methods: Plasma levels of total cholesterol, total triglyceride, and concentrations of cholesterol, triglyceride, and protein in LDL and HDL were measured in 12 preeclamptic women and 12 normal pregnant women. Oxidation of LDL or HDL was assessed by incubation with copper ions and evaluated by monitoring the kinetics of conjugated diene formation.
Results: The plasma levels of total triglyceride and concentration of LDL protein were significantly higher in pre-eclamptic women than in normals. Levels of HDL lipid did not differ significantly. Analysis of kinetics of conjugated diene production showed a significantly shorter lag time for LDL (83.1 ± 5.5 minutes versus 67.4 ± 10.2 minutes, P < .001) and HDL (76.9 ± 7.3 minutes versus 59.5 ± 9.2 minutes, P < .001) and a significantly higher oxidation rate for LDL (3.6 ± 0.4 nmol/minutes/mg LDL versus 4.4 ± 1.0 nmol/minutes/mg LDL, P < .05) in preeclamptic women.
Conclusion: Low-density lipoprotein and HDL particles were more susceptible to oxidative modification, and plasma concentration of LDL particles, but not of HDL particles, was increased in preeclampsia.
The vascular endothelium might contribute to regulation of vascular smooth muscle tone by producing such vasoconstrictors as endothelin-1 and thromboxane, and such vasodilators as prostacyclin and nitric oxide. Endothelial cell injury and altered endothelial function are important in the pathogenesis of pre-eclampsia.1 Serum lipids have a direct effect on endothelial function.2 Abnormal serum lipid profiles are associated with endothelial dysfunction.3 The plasma levels of cholesterol and triglyceride show an increase toward the term of normal pregnancy.4 The plasma concentrations of low-density lipoprotein (LDL)5 and very low-density lipoprotein particles2 are reportedly increased in women with preeclampsia compared with women with uncomplicated pregnancies.
Low-density lipoprotein is oxidized in the intimal spaces of arteries by oxygen free radicals. The oxidative modification of LDL increases its uptake by macrophages through scavenger receptors that are not down-regulated. Those macrophages accumulate large amounts of cholesterol and develop into foam cells in atherosclerotic lesions.6 The oxidatively modified LDL impairs endothelial cells by stimulating the vascular cell adhesion molecule-1,7 stimulating neutrophil adhesion receptors (CD11b/CD18),8 inhibiting endothelial prostacyclin and nitric oxide synthesis,9 and increasing endothelin production.9 Therefore, the oxidative modification of LDL might be involved in the impairment of vascular endothelial cells. In preeclampsia, decidual vessels show fibrinoid necrosis of the vascular wall and focal accumulation of lipid-laden macrophages, similar to atherosclerosis.1 Lipid peroxidation is enhanced in preeclamptic women.10 It is likely that LDL particles in preeclampsia are readily oxidized, and that the oxidized LDL contributes to the vascular endothelial damage.
High-density lipoprotein (HDL) enhances the efflux of cholesterol from macrophage-derived foam cells and decreases the amount of cholesteryl ester in the cells. However, HDL and LDL are oxidized in the subendothelial space. Oxidized HDL shows a lesser effect on the decrease of cholesteryl ester in foam cells.11 Therefore, the oxidative modification of HDL might be important in the development of atherosclerosis.
The present study investigated the susceptibility of LDL and HDL particles to oxidative modification in women with preeclampsia.
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Materials and Methods |
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Blood samples were collected from subjects between 8:00 AM and 10:00 AM after a 12-hour fast, with tubes that contained 1 mg/mL ethylenediaminetetra-acetic acid (EDTA) and centrifuged immediately at 1500 g for 20 minutes at 4C. None of the women were in labor at blood sampling. Low-density lipoprotein (density: 1.019–1.063) and HDL (density: 1.063–1.21) were subsequently fractionated from freshly drawn (within 24 hours) plasma samples by ultracentrifugation according to the method of Havel et al.14 The levels of total cholesterol and total triglyceride in plasma, and the levels of cholesterol and triglyceride in LDL and HDL subclasses, were measured enzymatically.15 Protein concentration was determined by the method of Lowry et al.16 To remove the EDTA, the isolated LDL and HDL fractions were dialyzed against 30 mM sodium phosphate buffer containing 150 mM NaCl, which was made oxygen free by vacuum degassing, followed by purging with nitrogen in the dark at 4C for 48 hours. The buffer was changed after 24 hours’ dialysis.
The EDTA-free dialyzed LDL and HDL fraction was diluted with dialysis buffer to a final concentration of 200 µg/mL. Oxidation was initiated by the addition of 2.0 µM CuSO4. The kinetics of the formation of conjugated dienes was determined by monitoring the change in the absorbance at 234 nm on a Beckman Model DU 640 spectrophotometer equipped with a 12-position automatic sample changer. Absorbance at 234 nm was recorded at 37C every 3 minutes for 4 hours. The lag phase, propagation phase, and decomposition phase were determined as previously described.17 Lag time was defined as the interval between the addition of CuSO4 and the intercept of the tangent of the slope of the absorbance curve with the time-scale axis during the propagation phase. The maximal oxidation rate was calculated from the slope of the tangent, using a molar extinction coefficient for conjugated dienes of 
234 = 29,500/mol/cm, and expressed as nanomoles of diene formed per minute per milligram of LDL or HDL protein. Amount of dienes was determined from the absorbance curve as the absorbance at the beginning of the decomposition phase minus the absorbance at the start of the lag phase. The corresponding amount of dienes was calculated as described for the oxidation rate.17
Data are expressed as the mean ± standard deviation (SD). Differences in characteristics, lipid levels, lag time, oxidation rate, and amount of conjugated dienes were analyzed by Student unpaired t test when there was normal distribution or Mann-Whitney test when parameters were not distributed normally. Differences in parity were evaluated by Fisher exact test. Regression lines were determined by the least squares method. A level of P < .05 was accepted as statistically significant.
By sample size calculations, 24 participants were needed to detect a 1.0-minute difference in lag time, given a 10% dropout rate, power of 90%, and 
= 0.05; SD for paired difference was 1.0.
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). The plasma level of triglyceride and the concentration of LDL protein were significantly greater in preeclamptic subjects, but the levels of other lipids did not differ significantly between groups (Table 2). The ratio of LDL triglyceride to LDL cholesterol was significantly higher in preeclamptic than control women (0.80 ± 0.40 versus 0.53 ± 0.18, P < .05). The plasma level of triglyceride correlated positively with the ratio of LDL triglyceride/cholesterol (y = 153.7x + 163.2, r = 0.58, P < .01). The kinetics of CuCO4-induced formation of conjugated dienes revealed a significantly shortened lag time for LDL and HDL particles, and a significantly greater oxidation rate of LDL, in women with preeclampsia. There was no significant difference in the oxidation rate of HDL. The groups did not differ in total amount of conjugated dienes formed in LDL and HDL particles (Table 3).
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Discussion |
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Low-density lipoprotein particles that accumulate in the plasma might infiltrate the intimal space of arteries to be oxidized by oxygen free radicals. Biologic oxidative modification can be mimicked by incubating LDL in a cell-free buffer with copper ions.17 In the present study, LDL or HDL was oxidized by incubation with Cu++ ions. Kinetic analysis of conjugated diene production showed that the lag time of that reaction, which indicates the intrinsic antioxidant activity of LDL particles, was shortened, consistent with a previous report,19 and the oxidation rate, which indicates the breakdown rate of polyunsaturated fatty acids, was greater in the preeclamptic subjects versus controls. That indicates that the LDL particles in preeclamptic subjects might have an increased susceptibility to oxidative modification. Antibodies to oxidized LDL have been reported increased in women with preeclampsia.20 Oxidatively modified LDL particles directly impair the vascular endothelial cells and the formation of foam cells. Thus, in preeclampsia, the increased plasma concentration of LDL particles might be oxidized easily in the intimal space, leading to foam-cell formation and vascular endothelial damage.
Small, dense LDL particles reportedly associated with increased risk of atherosclerosis.21 Small, dense LDL has a low affinity for hepatic LDL receptors, thus is more likely to accumulate in the bloodstream.22 Small LDL particles also are more susceptible to oxidative modification, which is an initial step in the atherosclerotic process.23 Elevated plasma levels of triglyceride might reduce the size of LDL particles in women with pre-eclampsia.12 We reported that in postmenopausal women the estrogen-induced increase in the plasma level of triglyceride produces small, triglyceride-rich, cholesterol-poor LDL particles.24 The present study found increased concentration of LDL-protein, but no significant difference in the concentration of LDL-cholesterol, in women with preeclampsia. Although we did not measure the size of the LDL particles, those results can be accounted for in part by an increased number of small LDL particles containing reduced lipid levels in those preeclamptic subjects. That concept is supported by the observation that the smaller, denser LDL particles contain a decreased level of cholesterol.22 In the present study, the ratio of LDL-triglyceride/cholesterol was significantly greater in preeclampsia and the plasma triglyceride level was correlated positively with the ratio of LDL-triglyceride/cholesterol. Those results suggest that an increased plasma level of triglyceride might produce small triglyceride-rich, cholesterol-poor LDL particles in preeclamptic subjects.
High-density lipoprotein, a protective factor against atherosclerosis, also is oxidized in the subendothelial space. Oxidized HDL is not taken up by the macrophages and does not lead to formation of foam cells. However, the reverse cholesterol transport system is impaired by oxidative modification.11 The plasma level of HDL cholesterol is reportedly reduced in preeclampsia.25 Although the mean concentrations of HDL lipids and HDL protein were lower in preeclamptic subjects, the differences were not statistically significant. Our results indicated that plasma concentrations of HDL particles did not differ between groups. The lag time for HDL was shortened in the preeclamptic subjects, whereas oxidation rates did not differ. That indicates that HDL particles in preeclampsia are more susceptible to oxidative modification but that the breakdown rate of polyunsaturated fatty acids might be unaffected.
Preeclamptic subjects showed an elevation of lipid peroxidation products.10 Lipid peroxidation is closely linked to antioxidants and polyunsaturated fatty acids present in lipoprotein particles. The amount of conjugated diene formed in LDL and HDL particles did not differ between groups, indicating that the concentration of polyunsaturated fatty acids in both particles was not altered in preeclampsia. However, the plasma level of antioxidants such as vitamin E is reportedly decreased in preeclampsia.10 Therefore, enhanced oxidative stress in preeclamptic subjects might reduce the concentration of endogenous antioxidants in lipoproteins, resulting in an increased susceptibility of LDL and HDL to oxidative modification. We showed that in postmenopausal women, the administration of melatonin, a powerful scavenger of oxygen free radicals by quenching the hydroxyl radicals and possibly the peroxyl radicals, reduces the susceptibility of LDL to oxidative modification.26 The administration of an antioxidant such as melatonin might affect favorably the susceptibility of LDL and HDL to oxidative modification and might reduce vascular endothelial damage in preeclamptic subjects. Further clinical studies are needed to clarify those issues.
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Footnotes |
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Received November 16, 1999. Received in revised form February 7, 2000. Accepted February 25, 2000.
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References |
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26. Wakatsuki A, Okatani Y, Ikenoue N, Izumiya C, Kaneda C. Melatonin inhibits oxidative modification of low-density lipoprotein particles in normolipidemic postmenopausal women. J Pineal Res. In press.
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