ࡱ> bdaxIfbjbj[][]>>>>8v$>%bb(####=#$J%$&(nn%Qn%? bl:#r#:#g#f,# D Ts*>>K#evaluatIon of the Placental lIpId peroxIdes, glutathIon peroxIdes actIvITIes AND THEIR RELATIONSHIP WITH SERUM LIPOPOPROTEINS In preeclamptIc and eclamptIc pregnancIes gkhan BAYHAN, Y1ld1z ATAMER, Ayta ATAMER, Yksel KOY00T Department of 1Obstetrics and Gynecology, 2Biochemistry, 3Internal Medicine, 4Physiology. Medical Faculty, Dicle University. Diyarbakir-Turkey Correspondence Address: Gkhan BAYHAN M.D., Assistant Professor of Ob./Gyn., Dicle University, School of Medicine Diyarbak1r, TURKEY Tlf: 00-90-412-2488430 E-Mail: bayhan@tr-net.net.tr SUMMARY Placental production of lipid peroxides (LPO) and levels of serum lipoproteins increase abnormally in preeclampsia (PE) and eclampsia (E). The cause of it is not known, but if placental antioxidant enzymes are deficient, lipid peroxides will increase. This study has examined any evidence of increased placental lipid peroxidation and accumulation of malonydialdehyde (MDA, an end product of LPO) in preeclamptic or eclamptic women and any relationship between the elevated MDA or lipoproteins and lower antioxidants in healthy pregnant women. Placental tissues and blood samples were obtained from normal (n=44), preeclamptic (n=27), eclamptic (n=18) pregnancies immediately after delivery. The levels of triglyceride (TG), total cholesterol (T.Chol), high density lipoprotein-cholesterol (HDL-C) and placental LPO, glutathione peroxidase (GSH-Px), glutathione (GSH) were measured seperately. GSH-Px activity and the levels of glutathione (GSH) were significantly lower in preeclamptic and eclamptic pregnancies than in normal placentas (p<0.0001). Lipid peroxides were significantly higher in the placentas of preeclamptic and eclamptic pregnancies than those of normal subjects (p<0.0001). The levels of serum TG were significantly higher in the PE and E group than those in the control group (p<0.0001). The levels of HDL in preeclamptic and eclamptic patients were significantly lower than those of control group (p<0.001), but the levels of LDL did not show a significant difference between the groups (p>0.05). The levels of serum T.Chol in preeclamptic and eclamptic pregnancies were significantly higher than those in the control group (p<0.0001). In preeclamptic group was found no correlation between the level of placental MDA and serum lipoproteins. However, in eclamptic group a significant positive correlation was noted between the appearance of placental MDA and the level of serum trigliyceride (p<0.001). Also, a negative correlation was obtained between the level of serum HDL and placental MDA in eclamptic group (p<0.02). Key words: Preeclampsia, eclampsia, lipid peroxides, glutathione, glutathione peroxides, lipoproteins, placenta. INTRODUCTION It has been recently hypothesized that in preeclamptic and eclamptic pregnancies a placental oxidant-antioxidant imbalance might cause the release of lipoperoxidant products into the circulation, with subsequent damage to endothelial cell membranes (1). It is clear that within the placenta, there is an increase in tumor necrosis factor-alpha (TNF-alpha) and lipid peroxide production. These changes are associated with a reduction in various placental antioxidants. This suggests there may be a failure of the normal fetal protection systems. Stimulated monocytes produce free radicals, which can cause oxidative damage. Maternal cells protect themselves with both plasma and intracellular antioxidants. Changes in membrane oxidation can lead to changes in the membrane stability (2). In preeclampsia, antioxidant activities of maternal plasma and placenta are significantly reduced in comparison with activity levels in normal pregnancies (1,19,20), thus contributing to the increase of placental and circulating levels of lipid peroxides (8). Glutathione peroxidase is one of the primary antioxidants that is present in tissues and that limits the amount of lipid peroxides (6,7). Glutathione peroxidase uses glutathione as its cofactor is to convert lipid peroxides into relatively harmless hydroxylated fatty acids, water, and glutathione disulfide. Therefore, if glutathione peroxidase activity is deficient, lipid peroxides can increase in the tissues (9). Changes in lipoprotein metabolism, namely very low density lipoproteins (VLDL) and oxidized low density lipoproteins (LDL) concentrations in plasma could participate in endothelial dysfunctions observed during preeclampsia (10). This study was performed to determine (1) whether the level of glutathione peroxidase and glutathione activities in placental tissue are deficient in women with preeclampsia and eclampsia, (2) whether this deficiency is associated with increased levels of lipid peroxides and (3) whether inhibition of glutathione peroxidase activity will result in increased placental production of lipid peroxides and lipoproteins. MATERIALS AND METHODS This study was conducted between April 1997 and February 1998 in the Department of Gynaecology and Obstetrics, Dicle University Faculty of Medicine. Forty-four healthy pregnant women were included in the study as a control group, considering their ages ranging from 20 to 40 years with pregnancy from 24 to 40 weeks, 27 patients with preeclampsia (from 19 to 45 years of age with pregnancy from 24 to 41 weeks) and 18 patients with eclampsia aged from 18 to 40 years with pregnancy of 29 to 40 weeks were carefully chosen. Patients with preeclampsia were defined on the basis of the following clinical and laboratory criteria: Systolic blood pressure > 140 mm Hg and diastolic blood pressure > 90 mm Hg noted on at least two occasions 6 hour apart, no fundoscopic findings with hypertensive rethinopathy, proteinuria levels > 300 mg/dl found in at least two random specimens 6 hour apart. Eclampsia was diagnosed on the development of convulsion with the clinical signs and symptoms consisted with preeclampsia. Forty-four patients in the second and third trimester without maternal and fetal complications during the pregnancy period were selected as control group. Pregnants who lacked these criteria were excluded. Gestational age was defined based on last menstrual period and ultrasonography. Furthermore, all patients were consulted by internal medicine department. None of the women had received antihypertensive medication until the study samples were taken. 44 control, 27 preeclamptic and 18 eclamptic women were evaluated. Placental tissue pieces (2 to 3 g each) taken from 44 normal and 27 preeclamptic women and 18 eclamptic women between 35 and 40 weeks gestation were immediately frozen in liquid nitrogen after delivery. There were thirty-two vaginal deliveries and twelve cesarean sections in the normal group and 20 vaginal deliveries and seven cesarean sections in the preeclamptic group and 14 vaginal deliveries and four cesarean sections in the eclamptic group. Tissues were strored at 80 C until assigned. Placental tissue homogenates were prepared as follows. Tissue pieces were ground up in a Waring blender with liquid nitrogen, then 1 gr of frozen tissue from each placenta was weighed and homogenized with 9.0 ml of 1.15 % potassium chlaride buffer. Aliquots of homogenates were then used for the analysis of glutathione peroxidase activity, glutathione and lipid peroxides. Placental malondialdehyde (MDA) level, which is the end-product of lipid peroxidation, was measured by Ohkawas thiobarbituric acid (TBA) method (11). In the measurements, the results were expressed as nmol MDA/g tissue by using molar extinction a coefficient (1.56 x 105 M-1 x cm-1 ). Placental GSH levels were measured by the dithio nitrobenzen (DTNB) method described by Ellmen (12), and the results were expressed micromol GSH/g tissue. Placental GSH-Px activity was measured by the method of Hafemen, Sande and Hoekstra (13). When the decrease of log (GSH) per minute in enzymatic reaction was substracted from the decrease of log (GSH) per minute in non-enzymatic reaction, each 0.001 unit reduction calculated was identified as 1 enzyme unit, and results were pointed out as Unit/100 mg tissue. Blood samples were obtained from the eclamptic and preeclamptic pregnancies and healthy pregnancies under overnight fasting conditions. After the fractionation of the sera was made, total cholesterol (Tchol), triglycerid (TG) and High density lipoprotein-cholesterol (HDL-C) were measured on the same day. T. Chol, TG and HDL-C levels were assayed by enzymatic calorimetric in Beckmen Synchron Cx-5 autoanalyzer. LDL-C levels were calculated by using the formula of Friedewald (14). In the statistical evaluation of the results, one way variance analysis was used and Tukey HSD was used as a post hoc. Analyses were performed using SPSS software (Statistical Package for the Social Sciences, version 6.0). Correlation between different markers was determined. RESULTS The description of the group was given in Table I. The mean age of eclamptic women was significantly different, when compared with those of preeclamptic and control groups (p<0.001). There was no statistical difference in mean gestational weeks between all three groups (p<0.05). The mean systolic and diastolic blood pressure of those was found statistically significant compared to the control group (p<0.001). The tissue levels of glutathione peroxidase activity were significantly lower in preeclamptic and eclamptic placentas than in normal placentas (p<0.0001). In contrast the tissue levels of lipid peroxides were significantly higher in preeclamptic and eclamptic placentas than in normal placentas (p<0.0001). (Table II). Similar to GSH-Px levels, glutathione levels were also significantly lower in preeclamptic and eclamptic placentas compared with normal placentas (p<0.0001) (Table II). Serum T.Chol and TG levels in eclamptic and preeclamptic groups were found higher than those of control group. The differences were significant (p<0.0001) (Table III). Levels of serum HDL in preeclamptic and eclamptic groups were significantly lower than those of control group (p<0.0001), whereas LDL levels were not different between the groups (p>0.05) (Table III). The ratios of HDL cholesterol to LDL cholesterol were significantly decreased with respect to controls in women with preeclampsia and eclampsia (p<0.0001). However, the ratios of MDA to LDL cholesterol and MDA to HDL cholesterol in preeclamptic and eclamptic group were significantly high when compared to the ratios of the control group (p<0.0001) (Table III). In eclamptic group a significant positive correlation was noted between the appearance of placental MDA and the level of serum trygliceride (p<0.001). To exclude the possibility that the enhanced lipid concentration in preeclampsia and eclampsia could be responsible for the results, correlations between lipid peroxidation (MDA) and lipoproteins were determined. A negative correlation was obtained between the level of serum HDL and placental MDA (p<0.02). However in eclamptic group the placental MDA did not show any correlation to cholesterol, nor was there any correlation between placental MDA and the level of LDL. In preeclamptic group was found no correlation between the level of placental MDA and serum lipoproteins. DISCUSSION Increasing evidence supports the role of abnormal lipid metabolism and circulating modified lipids in the pathophysiologic mechanisms of preeclampsia or eclampsia (4,9,15,17). In particular, lipid peroxides and their related free radicals have been implicated in the pathogenesis of placental dysfunction in preeclampsia (6,9,15,17). The concentration and production of lipid peroxides by placental tissue from women with preeclampsia is also significantly elevated (4,16,17,18). In this study we have demonstrated that the tissue levels of GSH-Px activity and GSH levels were significantly lower in placentas obtained from women with preeclampsia and eclampsia than in placentas obtained from women with normal pregnancies and we have clarified that there were changes in lipid content and in placental lipid peroxide levels of serum lipoprotein fractions in preeclamptic and eclamptic pregnant subjects. Among the results obtained it was found that LPO levels in the HDL fraction of preeclamptic or eclamptic subjects singificantly decreased as compared with the level of normal pregnancies, and that the ratios of lipid peroxide levels to the amounts of lipids (MDA/LDL-C and MDA/LDL-C) in same fraction of preeclamptic-eclamptic subjects were also significantly higher than those of normal pregnant women. GSH also has important roles in xenobiotic metabolism and leukotriene synthesis and is found at millimoler concentrations in all human cells (19). Glutathione peroxidase is one of the primary antioxidants that is present in tissues and that limits the amount of lipid peroxides (9). Because antioxidant defences are not completely efficient, increased free-radical formation in the body is likely to increase damage (20). Therefore, if glutathione peroxidase activity and GSH levels are deficient, lipid peroxides can increase in the tissues (9,16). The decreased levels of glutathione peroxidase activity were associated with significantly increased levels of lipid peroxides (9). Also in this study placental LPO increased significantly, and GSH and GSH-Px in placenta decreased significantly in women with preeclampsia and eclampsia compared with controls. Our results were consistent with the previous reports (2,3,21,22). The placenta acts as a source of lipid peroxides and these changes are a primary event in the pathophysiology of endothelial lesions (4). Important indirect evidence supports the notion that elevation in lipid peroxide levels is not merely a consequence of endothelial lesions (5). In a recent report endothelial stimulating activity of plasma from women with preeclampsia or eclampsia appeared to depend on the lipid fraction of plasma, subsequent lipoprotein (LDL) and very low density lipoprotein fractions (VLDL) (10). It is not known whether smaller subfractions of LDL are further increased in women with preeclampsia or what the level of oxidative modification of the different subfractions in these women is. Lipoproteins might undergo intensive oxidative modifications as they interact with vascular endothelium of an ischemic and/or inflammatory maternal-fetal interface, initiating and propagating the lipid peroxidation process and leading to generalized endothelial dysfunction (4,23,24). Preeclampsia or eclampsia is further characterized by an abnormal elevation in triglyceride levels beyond the physiologic increase of normal pregnancy, which is generally regarded as further evidence of the implication of lipid disturbances in its pathophysiologic mechanisms (4,15). Marked hypertriglyceridemia, Potter and Nestel observed in preeclamptic subjects, is mainly due to increased TG in the VLDL and LDL fractions (25). However, in this study serum TG and T.Chol were significantly higher in preeclamptic or eclamptic than in normal placentas, whereas LDL-C did not differ from those observed in the group with preeclampsia or eclampsia and control groups. In addition, levels of serum HDL-C and ratio of HDL-C/LDL-C were significantly lower in preeclamptic or eclamptic than in normal pregnancies. In fact, the ratio of HDL-C to LDL-C, one of the risk factors of vascular disease, significantly decreased in preeclamptic or eclamptic women (15). In the present study we observed that the T.Chol and TG levels were elevated in preeclamptic pregnant subjects when compared with normal pregnants, and that the level was further elevated in eclamptic subjects in accordance with the previous paper (4,15,25). Consequently, it might be possible to consider that lipid peroxides produced in cell membrane are transferred to the HDL fraction together with lipids and circulate in the blood stream leading to various diseases such as preeclampsia-eclampsia. On the other hand, high lipid peroxide levels in preeclamptic-eclamptic subjects were mainly ascribed to the increase in the amount of lipids, especially to that of triglyceride in VLDL. Accordingly, the increase of LOP levels in preeclamptic-eclamptic subjects were probably due to the increased TG level in serum with any reflection of membrane damage. In preeclamptic-eclamptic subjects, however, a further change in HDL fraction occurs, as mentioned above (15,25). To exclude the possibility that the enhanced lipid concentration in preeclampsia and eclampsia could be responsible for the results, the correlation between placental lipid peroxidation and serum lipoproteins was determined. In eclamptic group a significant positive correlation was noted between the appearance of placental MDA and the level of serum TG (r=0.4489, p<0.001). Also, a negative correlation was obtained between the level of serum HDL and placental MDA (r= -0.464, p<0.02). However, the placental MDA did not show a correlation to T.Chol, nor was there a correlation between placental MDA and the level of LDL. This correlates well with the notion that the placenta acts as a source of lipid peroxides and that these changes are a primary event in the pathophysiology of endothelial lesions. In preeclamptic group there were found no correlation between the level of placental MDA and serum lipoproteins. Acknowledgment We would like to thank Aytekin SIR for his helpful assistance in statistical methods. REFERENCES Cester N, Staffolani R, Rabini RA, Magnanelli R, Salvolini E, Glassi R, Mazzanti L, romanini C. Pregnancy induced hypertension: a role for peroxidation in microvillus plasma membranes. Mol-Cell-Biochem. 1994; 131: 151-5. Walker JJ. Antioxidants and inflammatory cell response in preeclampsia. Semin Reprod Endocrinol. 1988; 16: 47-55. Cueto SM, Romney AD, Wang Y, Walsh SW. Beta-Carotene attenuates peroxide-induced vasoconstriction in the human placenta. J Soc Gynecol Investig. 1997; 4: 64-71. Gratacos E, Casals E, Deulofeu R, Carrarach V, Alonso P and Fortuny a. Lipid peroxide and vitamin E patterns in pregnant women with different types of hypertension in pregnancy. Am J Obstet Gynecol. 1998; 178: 1072-6. Davidge ST. Oxidative stress and altered endothelial cell function in preeclampsia. Semin Reprod Gndocrinol. 1998; 16: 65-73. Morikawa S, Kurauchi O, Tanaka M, Yoneda M, Uchida K, Itakura A, Furugori K, Mizutani S, Tomoda Y. Increased mitochondrial damage by lipid peroxidation in trophoblast cells of preeclamptic placentas. Biochem Mol Biol Int. 1997; 41: 767-75. Shaarawy M, Areff A, Salem ME, Sheiba M. Radical scavenging antioxidants in preeclampsia and eclampsia. Int J Gynecol Obstet. 1998; 60: 123-8. Tabacova S, Balabaeva L. Maternal exposure to exogenous nitrogen compounds and complications of pregnnancy. Arc of Env Health. 1997; 52: 341-7. Walsh SW, Wang Y. Deficient glutathione peroxidase activity in preeclampsia is associated with increased placental production of thromboxane and lipid peroxides. Am J obstet gynecol. 1993; 169: 1456-61. Lefevre G, Berkane N, Uzan S, Etienne J. Preeclampsia and oxygenated free radicals Ann Biol Clin. 1997; 55: 443-50. Ohkawa H, Ohishi N and Yagi N. Assay for lipid peroxidase in animal tissues by thiobarbituric acid reaction. Anal biochem 1979; 95: 351-8. Ellman Gl. Tissue sulfhydryl groups. Arch Biolchem Biophys 1959; 82: 70-7. Hafeman DG, Sunde RA and Hoekstra WG. Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J Nutr 1974; 104: 580-7. Friedewald WT, Levy R, Frederickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499-502. Maseki M, Nishigaki I, Hagihara M, Tomoda Y and Yagi K. Lipid peroxide levels and lipid content of serum lipiprotein fractions of pregnant subjects with or without preeclampsia. Clin Chim Acta 1981; 115: 155-61. Wang Y, Walsh SW. Antioxidant activities and mRNA expression of superoxide dismutase, catalase and glutathione peroxidase in normal and preeclamptic placentas. J Soc Gynecol Investig. 1996; 3: 174-84. Jain SK, Wise R. Relationship between elevated lipid peroxides, vitamin E deficiency and hypertension in preeclampsia. Mol-Cell-Biochem. 1995; 151: 33-8. Liang X, Lin Y, Cheng Y. Changes in plasma endotehlin-1 and lipid peroxidate levels and amount of superoxide dismutase in red blood cell in patients with pregnancy-induced hypertension. Chung-Hua-Fu-Chan KoTsa-Chih 1996; 31: 220-2. Chance B, Sies H, Boveris A. Hydropero_ide metabolism in mammalion organs Physiol Rev. 1979; 59: 527-605. Sarafian TA, Bredesen DE. Is apoptosis metiated by reactive oxygen species? Free Radic Res. 1994; 20: 1-6. Chen G, Wilson R, Cumming G, Walker JJ, Smith WE and McKillop JH. Prostacyclin, thromboxane and antioxidant levels in pregnancy-induced hypertension. Eur J Obstet Gynecol Reprod Biol 1993; 50: 243-250. Jendryczko A, Tomala J. Decreased activity oxidoreductases in erythrocytes and blood platelets from venous and umbilical blood of women with pregnancy- induced hypertension. Ginekol Pol. 1995; 66: 652-9. McNamara JR, Jenner JL, Li Z, Wilson PWF, Schaefer EJ. Change in LDL particle size is associated with change in plasma triglyceride concentration. Arterioscler Thromb. 1992; 12: 1284-90. Silliman K, Shore V, Forte TM. Hypertriglyceridemia during late pregnancy is associated with the formation of small dense low density lipoproteins and the presence of large buoyant high-density lipoproteins. Metabolism 1994; 43: 1035-41. Potter JM and Nestel PJ. The hypertlipidemia of pregnancy in normal and complicated pregnancies. Am J Obstet Gynecol. 1979; 15: 165-70. Table I. Description of the groups NP (n=44)PE (n=27)E (n=18)Age (years)29.9 ( 5.729.2 ( 7.023.6 ( 6.4Nulliparous (%)546570Systolic blood pressure (mmHg)119.1 (8.2150.7 ( 10.3147.7 (14.3Diastolic blood pressure (mmHg)82.6 ( 7.8103.3 ( 10.396.1 (8.4Gestational age at delivery (weeks)35.8 ( 4.435.5 ( 5.134.9 ( 3.8Proteinuria (g/day)06.7 ( 1.38.5 ( 6.4Birthweight (g)3512 (5132851 (5582126 ( 718 Table II. Statistical significance of placental MDA and GSH-Px activity and the level of GSH in preeclamptic, eclamptic and control pregnancies. VariablesGroupsX ( SDpMDAControl group Preeclampsia Eclampsia105.51 ( 4.27 133.72 ( 6.44 145.66 ( 5.85 <0.0001 <0.0001GSHControl group Preeclampsia Eclampsia5.98 ( 0.23 4.82 ( 0.29 4.36 ( 0.32 <0.0001 <0.0001GSH-PxControl group Preeclampsia Eclampsia514.72 ( 3.56 501.17 ( 5.59 493.79 ( 9.52 <0.0001 <0.0001 Table III.The serum level of lipoproteins and ratios of MDA/LDL-C, MDA/HDL-C and HDL-C/LDL-C in preeclamptic, eclamptic and control pregnancies. VariablesGroupsX ( SDpT.KolControl group Preeclampsia Eclampsia189.95 ( 21.27 226.81 ( 69.14 214.55 ( 42.80 <0.003 <0.003TGControl group Preeclampsia Eclampsia133.22 ( 23.54 277.18 ( 120.64 299.55 ( 104.44 <0.0001 <0.0001HDLControl group Preeclampsia Eclampsia40.47 ( 2.59 28.48 ( 3.97 26.38 ( 3.97 <0.0001 <0.0001LDLControl group Preeclampsia Eclampsia122.83 ( 18.44 142.89 ( 57.53 128.25 ( 34.97 >0.05 >0.05HDL-C/LDL-CControl group Preeclampsia Eclampsia0.336 ( 0.551 0.233 ( 0.107 0.242 ( 0.170 <0.0001 <0.0001MDA/LDL-CControl group Preeclampsia Eclampsia0.877 ( 0.138 1.099 ( 0.507 1.300 ( 0.721 <0.0002 <0.0002MDA/HDL-CControl group Preeclampsia Eclampsia2.618 ( 0.220 4.806 ( 0.860 5.630 ( 0.813 <0.0001 <0.0001  Figure I. The level of MDA in preeclamptic, eclamptic and control pregnancies  Figure II.The level of GSH in preeclamptic, eclamptic and control pregnancies  Figure III.The level of GSH-Px in preeclamptic, eclamptic and control pregnancies. 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