Increased susceptibility to low density lipoprotein oxidation in women with a history of pre-eclampsia

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1 BJOG: an International Journal of Obstetrics and Gynaecology April 2003, Vol. 110, pp Increased susceptibility to low density lipoprotein oxidation in women with a history of pre-eclampsia Eduard Gratacós a,b, *, Elena Casals b, Olga Gómez b, Elisa Llurba a, Imma Mercader b, Vicenç Cararach b, Lluís Cabero a Objectives To evaluate the susceptibility to oxidation of low density lipoprotein (LDL) in women with a history of pre-eclampsia. Design A case control study. Setting The departments of obstetrics and gynaecology at two university teaching hospitals. Population Women delivering one to three years before enrolment, 35 who were diagnosed with severe preeclampsia and 35 controls matched for age, body mass index (BMI), smoking and parity. Methods Plasma samples were analysed for total cholesterol, high density lipoprotein (HDL) cholesterol, LDL cholesterol, triglycerides and lipoprotein A. The in vitro susceptibility to oxidation of LDL was measured and expressed in minutes (lag time). Results are expressed as mean and standard deviation. Main outcome measures Serum lipid profile and in vitro susceptibility to oxidation of LDL. Results Mean LDL cholesterol (116 [37] vs 98 [20] mg/dl, P < 0.05) and trygliceride (112 [56] vs 78 [38] mg/dl, P < 0.05) levels were significantly higher in the groups of women who had pre-eclampsia compared with controls. The rest of the measured lipid parameters were similar between the two study groups. The susceptibility to oxidation of LDL was also significantly higher in the pre-eclampsia group (lag time: 37.9 [8.4] vs 44.8 [9.1] minutes, P < 0.01). Conclusion Women with a history of pre-eclampsia have significant differences in lipid parameters and an increased susceptibility to lipoprotein oxidation when compared with women who had a normal pregnancy one to three years after delivery. INTRODUCTION Pre-eclampsia develops in about 2% of pregnancies and is still responsible for a significant proportion of perinatal and maternal morbidity and mortality 1. Pre-eclampsia has long known to be associated with abnormal placentation and impaired placental perfusion. However, other conditions characterised by poor placentation, such as intrauterine growth restriction, do not necessarily result in pre-eclampsia 2. This has lead to the growing concept that maternal predisposing factors must combine with the placental disorder to result in the pre-eclamptic maternal syndrome 3. In recent last years, several lines of evidence have suggested that part of this maternal predisposition a Fetal Medicine Unit, Department of Obstetrics and Gynaecology, Hospital Vall d Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain b Department of Obstetrics and Gynaecology, Hospital Clínic, Universitat de Barcelona, Barcelona, Spain * Correspondence: Dr E. Gratacós, Unitat de Medicina Fetal, Departament d Obstetrícia i Ginecologia, Hospital Materno-Infantil Vall d Hebron, Pg. Vall d Hebron , Barcelona, Spain. D RCOG 2003 BJOG: an International Journal of Obstetrics and Gynaecology doi: /s (03) could be explained by abnormal lipid metabolism. The levels of low density lipoprotein (LDL) cholesterol and triglycerides are increased in the non-pregnant state in women with past pre-eclampsia 4,5. The prevalence of lipid patterns commonly present in atherosclerosis is markedly increased in postmenopausal women with a history of eclampsia 6. Additionally, several gene mutations affecting lipoprotein lipase activity and associated with dyslipoproteinemia are more prevalent in women with pre-eclampsia 7. The association between dyslipemia and pre-eclampsia could be explained by endothelial dysfunction prior to pregnancy. Accumulating evidence suggests that increased LDL cholesterol and/or tryglicerides are associated with impaired endothelial function even in young healthy patients 8. An essential step to promote lipid-mediated endothelial dysfunction is the oxidative modification of LDL. Oxidatively modified LDL has been shown to markedly impair vasodilation and other endothelium-regulated processes 9. The susceptibility of LDL to oxidation can be reliably measured in vitro 10,11. This parameter is increased in chronic endothelial diseases, such as atherosclerosis or diabetic vasculopathy 8,9, and it correlates better with endothelial dysfunction than does cholesterol level 12. It is therefore commonly accepted as an indirect measurement of lipid abnormalities associated with endothelial dysfunction

2 SUSCEPTIBILITY TO LDL OXIDATION IN WOMEN WITH A HISTORY OF PRE-ECLAMPSIA 401 In this study, we tested whether women with a history of pre-eclampsia exhibited in the non-pregnant state an increased susceptibility to oxidation of LDL lipoproteins. We evaluated this and other lipid parameters one to three years after delivery in women who had presented with severe pre-eclampsia, as compared with women who had normal pregnancies. METHODS This was a case control study approved by the Institutional Ethical Committees, and verbal informed consent was obtained. Cases were selected among women delivering at any of the participating institutions from 1996 to Eligible cases were singleton deliveries with the diagnosis of severe pre-eclampsia. Women with any form of diabetes or other concurrent medical complication previous to or developing during pregnancy were not considered eligible for the study. The criteria for the definition of pre-eclampsia were those of the International Society for the Study of Hypertension in Pregnancy 13. Pre-eclampsia was diagnosed if a previously normotensive woman had two repeat (4 hours apart) diastolic blood pressure measurements of 90 mmhg or greater after the 20th week of gestation, along with proteinuria of more than 300 mg/l in 24 hours as measured quantitatively. Severe pre-eclampsia was established if any of the following was present: persistent diastolic blood pressure greater than 110 mmhg, platelet count <10 5 /ml, elevated transaminase levels, haemolysis or neurologic involvement. Women were identified from a pre-existing database of hypertensive complications in pregnancy, and in all cases, the diagnosis was reconfirmed after checking the clinical records. A total of 58 eligible women were identified, 51 were found and 49 agreed to participate. All women were interviewed using a structured questionnaire. Blood pressure was measured. The following exclusion criteria were used: (1) development of hypertension, diabetes or other chronic disease after the first pregnancy, (2) pregnancy and delivery less than six months before enrolment or (3) use of oral contraception. Sixteen cases were excluded because of one or more of these criteria, which left 35 women. Cases were matched one-to-one to controls, who were selected from a consecutive sample of women delivering at least one year before enrolment at any of the two institutions, and attending the outpatient clinics for routine gynaecologic checking. For each case, a healthy woman with a normal pregnancy history, matched for age (with standard deviation of 2 years), body mass index (BMI, weight/[height] 2 ) (with standard deviation of 2) and smoking status (yes/no) and without any of the exclusion criteria mentioned above was selected as a control. None of the cases or controls was using medication in the month before enrolment. A fasting blood sample between 8:00 and 10:00 a.m. was obtained for each woman and collected in Vacutainer tubes. Serum was separated by centrifugation, and samples were immediately stored at 80jC until assayed. Serum cholesterol and triglyceride levels were measured by enzymatic methods (Trinder, Bayer Diagnostics, Tarrytown, New York, USA) adapted to a Cobas Mira automated analyser (Hoffmann Larroche, Basel, Switzerland). Susceptibility to LDL oxidation was measured as previously described 14 according to the methods described by Esterbauer et at. 10 and Lunec 11. The method consists in measuring the formation of diene conjugates following exposure of LDL to copper. This requires monitoring with spectrophotometry the changes in absorbance at 274 nm. Briefly, LDL fractions were separated by ultracentrifugation, and samples were diluted with phosphate-buffered saline to result in a final concentration of 50 Ag protein LDL/mL. LDL was incubated at 37jC and the oxidation was initiated by the addition of 20 AL of freshly prepared 0.5 mm copper sulphate. Absorbance at 234 nm was recorded on a UV Vis Perkin-Elmer 550 spectrophotometer equipped with a Perkin-Elmer 561 recorder. Absorbance was set to zero in a first reading and the increment was recorded at 1 minute intervals. The formation of dienes and therefore the changes in absorbance follow a characteristic pattern: (1) a latency or lag phase, during which the formation of dienes is minimal, (2) a propagation phase characterised by a rapid increase in absorbance, reflecting rapid formation of dienes and (3) a decompensation phase, where the maximal concentration Table 1. Characteristics of study groups. Values given are mean [SD]. Pre-eclampsia Controls P Age at delivery (years) 29.4 [5.8] 28.5 [5.4] NS Age at enrolment (years) 31.3 [5.2] 31.0 [4.9] NS Parity 1.3 (0.2] 1.4 [0.2] NS BMI at enrolment (kg/m 2 ) 24.2 [2.8] 25.1 [3.3] NS Gestational age at delivery (weeks) 34.4 [3.2] 39.4 [1.6] < Mean arterial pressure* during pregnancy (higher recorded value) 136 [8] 88 [8] < Mean arterial pressure* at enrolment (mmhg) 96 [8] 90 [8] NS Birthweight (g) 2216 [690] 3177 [305] < * Mean arterial pressure ¼ diastolic blood pressure þ 1/3 (systolic diastolic blood pressure).

3 402 E. GRATACÓS ET AL. Table 2. Levels of serum cholesterol, triglycerides and lipoprotein A in study groups. Values given are mean [SD]. Pre-eclampsia Controls Total cholesterol (mg/dl) 191 [42] 174 [30] LDL cholesterol (mg/dl) 116 [37]* 98 [20] HDL cholesterol (mg/dl) 61 [18] 60 [21] Triglycerides (mg/dl) 112 [56]* 78 [38] Lipoprotein A (mg/dl) 28 [19] 28 [21] * Student s t test for paired samples, P < of dienes is reached and absorbance is stabilised again. The intra- and inter-assay variability was <10% for all the measurements. The following parameters were calculated: lag time, maximal oxidation rate and maximal concentration of conjugated dienes. Lag time (minutes) reflects the duration of the lag phase, which is the interval between zero time and the start of the propagation phase, as calculated by the intersection of the tangent of the slope of the absorbance curve during the propagation phase with the time-scale axis. Lag time is assumed to reflect the susceptibility or resistance of LDL to oxidation, and it is the most widely accepted and reliable marker of LDL oxidisability Maximal oxidation rate (nanomoles of dienes per milligram protein LDL per minute) was calculated from the slope of the absorbance curve during the propagation phase. Maximal concentration of conjugated dienes was expressed as nanomoles of dienes per milligram protein LDL. Lipoprotein A levels were measured immunochemically with a sandwich enzyme-linked immunosorbent assay that uses a monoclonal antibody to apolipoprotein A as the capture antibody, and levels are expressed as milligrams per deciliter. Data were analysed with the SPSS 10.0 statistical package (SPSS, Chicago, Illinois, USA). Since the distribution of values was normal, Student s t test for paired samples was used to evaluate the possible differences between the study groups. Fig. 1. Lag time (minutes) values for lipoprotein oxidation in women with a history of pre-eclampsia and controls. higher levels of triglycerides and LDL cholesterol (Table 2). There were no significant differences in total cholesterol or lipoprotein A between cases and controls. Values obtained for lipoprotein oxidation analysis are shown in Table 3. Lag time for LDL oxidation was significantly shorter in women with pre-eclampsia (Fig. 1). Oxidation rate (V max ) did not differ between cases and controls. The was a trend for higher values in the maximal concentration of conjugated dienes (C max ) in the group of pre-eclampsia, but the difference did not reach statistical significance. Finally, the potential effect of the time interval between delivery and plasma sampling was studied. Mean lag time values were similar among women studied between year one to two postdelivery and women studied beyond the second year postdelivery. RESULTS Clinical features of cases and controls are displayed in Table 1. Women with past pre-eclampsia had significantly Table 3. Susceptibility to LDL oxidation in cases and controls. Values given are mean [SD]. Pre-eclampsia Controls Lag time (minutes) 37.9 [8.4]* 44.8 [9.1] V max (Amol dienes/g LDL/minute) 31.8 [10.5] 33.5 [12.9] C max (Amol/mmol cholesterol) 884 [130] 790 [98] * Student s t test for paired samples, P < DISCUSSION This study provides evidence of an increased susceptibility to LDL oxidation in women with a history of preeclampsia. The susceptibility to oxidative modification is strongly associated with the size and density of LDL particles 15,16. Small, dense LDL particles demonstrate a greater susceptibility to oxidation than large and buoyant LDL 16, and therefore the predominance of this LDL subfraction is normally associated with an increased susceptibility to lipoprotein oxidation. The oxidative modification of small, dense LDL is commonly accepted to be an essential step in the genesis of lipid-mediated endothelial dysfunction 9,15,16. Oxidised LDL impairs endothelial function at various levels, including nitric oxide synthesis and

4 SUSCEPTIBILITY TO LDL OXIDATION IN WOMEN WITH A HISTORY OF PRE-ECLAMPSIA 403 destruction, platelet aggregability and expression of adhesion molecules 17. Pre-eclampsia is characteristically associated with hypertriglyceridemia, elevated lipid peroxides and a predominance of small, dense LDL 18,19. As recently reported, the susceptibility to LDL oxidation is increased during the acute phase of pre-eclampsia 20. These and other findings have led to the increasingly accepted notion that abnormal lipid metabolism is involved in the pathogenesis of endothelial dysfunction in pre-eclampsia 21. A more recent line of research further proposes that abnormal lipid metabolism could be a constitutional feature and therefore represent a predisposing factor to pre-eclampsia 4 7,22. The data described in this study support the notion that increased susceptibility to LDL oxidation could be a constitutional component in a proportion of women with a history of pre-eclampsia. About 40% (13/33) of cases demonstrated lag time values below the 2 SD of women with normal pregnancies one to three years after delivery. In the present study, cases and controls were matched for smoking status and BMI, which eliminates potential biases. On the other hand, the women were studied at least one year after delivery, which makes very unlikely the possibility that our observations could be explained by a persistence of pregnancy changes. Our data are in line with previous studies reporting the existence of abnormal lipid metabolism in women with a history of pre-eclampsia 4 6. Increased plasma levels of triglycerides and LDL cholesterol, together with reduced high density lipoprotein (HDL) cholesterol have been reported in two studies on pre-eclamptic women a few months after delivery 4,5. In another study, postmenopausal women with a history of eclampsia had an increased prevalence of dysliproteinemia as compared with age-matched controls with normal pregnancies 6. Altogether, these studies suggest that the lipid profile observed during the symptomatic phase of pre-eclampsia might persist as a constitutional feature in a proportion of patients, sharing important similarities with the atherogenic lipid phenotype 23. This lipid profile, characterised by hypertriglyceridemia, predominance of small, dense LDL and increased susceptibility to LDL oxidation is strongly associated to and constitutes a major risk factor for the development of atherosclerosis 9,14,15,23. The atherogenic lipid phenotype is not exclusive of atherosclerosis, but may be found in other dyslipemic conditions, such as diabetes mellitus and familial dyslipemias 23. The expression of this lipid pattern is to a large extent genetically determined 23, which further supports the existence of common aetiologic links between pre-eclampsia and atherosclerosis. The similarities between pre-eclampsia and atherosclerosis have been noted for more than two decades 24. The vascular lesion in the placenta of women with pre-eclampsia resembles the features of the atherosclerotic plaque 24. Preeclampsia and atherosclerosis are both endothelial diseases with an important involvement of lipid-mediated oxidative damage, and their lipid profiles are remarkably similar 21. Pre-eclampsia and atherosclerosis have also common risk factors, such as black race, obesity, hyperhomocysteinemia and diabetes 21. Recent evidence suggests that several mutations affecting the regulation of lipoprotein lipase are detected with an increased frequency in patients with preeclampsia 7. A genetic predisposition at the level of lipid metabolism is in agreement with the hypothesis of a multifactorial model to explain the maternal genetic contribution to pre-eclampsia 3,25. In summary, the findings reported in this article suggest that a proportion of women who develop pre-eclampsia have in the non-pregnant state an increased susceptibility to lipoprotein oxidation and a lipid profile that shares features with the atherogenic profile commonly observed in other dyslipemic conditions. This pattern could be a marker of genetically determined underlying disorders in lipid metabolism predisposing to oxidative stress and endothelial damage. Acknowledgements This study was supported by the Fondo de Investigaciones Sanitarias (Project 01/1397). References 1. Roberts JM, Lain KY. Obstetrics. Preterm birth and pre-eclampsia bad news and good news. Lancet 1998;352(Suppl 4):SIV Khong TY, De Wolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by preeclampsia and by small for gestational age infants. Br J Obstet Gynaecol 1986;93: Broughton Pipkin F. 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