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Lipid peroxide and vitamin E patterns in pregnant women with different types of hypertension in pregnancy
Lipid peroxide and vitamin E patterns in pregnant women with different types of hypertension in pregnancy Eduard Gratacós, MD,a Elena Casals, MD,b Ramón Deulofeu, MD,b Vicenç Cararach, MD,a Pedro L. Alonso, MD,c and Albert Fortuny, MDa Barcelona, Spain OBJECTIVES: We sought to evaluate the circulating levels of lipid peroxides and vitamin E and the placental levels of lipid peroxides in pregnant women with different types of hypertension. STUDY DESIGN: Lipid peroxides were measured in serum and placental tissue by the thiobarbituric acid method and high-pressure liquid chromatography, and vitamin E was measured by high-pressure liquid chromatography. The patients studied were 36 healthy pregnant women and 92 women with hypertension classified as having mild gestational hypertension (n = 28), severe gestational hypertension (n = 10), preeclampsia (n = 34), and chronic hypertension (n = 20). RESULTS: Lipid peroxides in serum and placental tissue were significantly increased, and vitamin E levels in serum were significantly decreased in women with severe gestational hypertension and preeclampsia compared with controls. The groups of mild gestational hypertension or chronic hypertension had similar values of lipid peroxides or vitamin E as controls. CONCLUSIONS: Our results suggest that the category of gestational hypertension may be composed of at least two entities with different pathophysiology and support the concept of nonproteinuric preeclampsia. (Am J Obstet Gynecol 1998;178:1072-6.)
Key words: Preeclampsia, gestational hypertension, lipid peroxides, vitamin E
Increasing evidence supports the role of abnormal lipid metabolism and circulating modified lipids in the pathophysiologic mechanisms of preeclampsia. In particular, lipid peroxides and blood oxidative imbalance are proposed as part of the cytotoxic mechanisms leading to endothelial cell injury.1 Lipid peroxides have been found to be elevated in the serum of women with preeclampsia compared with normal pregnant women,2, 3 and some correlation appears to be present between lipid peroxide levels and the severity of hypertension.4 In addition, plasma antioxidant activity5 and plasma levels of antioxidant vitamins (i.e., vitamins E and C) are low in these women.6 The concentration and production of lipid peroxides by placental tissue from women with preeclampsia is also significantly elevated. 7 However, these observations have not been evaluated in other forms of hypertension during pregnancy, such as gestational hypertension. From the Departament d’Obstetrícia i Ginecologia,a Servei de Bioquimica,b and Unitat d’Epidemiologia i Bioestadística, c Hospital Clinic de Barcelona, Universitat de Barcelona. Supported by grants from the Hospital Clínic and from the Fondo de Investigaciones Sanitarias FIS 96/1100. Received for publication July 18, 1997; revised November 14, 1997; accepted November 20, 1997. Reprint requests: Eduard Gratacós, MD, Department of Obstetrics and Gynecology, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/1/87751
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Pregnancy-induced hypertension complicates 5% to 10% of pregnancies, but the prognosis for the fetus and mother depends on the hypertensive syndrome. General agreement exists that a distinction must be made between preeclampsia and gestational hypertension, a usually more benign form of hypertension in pregnancy.8, 9 However, certain disagreement exists among different classifications as to the definition of preeclampsia, and some classifications require the presence of proteinuria,8, 9 whereas others do not.10 Indeed, the presence of proteinuria is a strong indicator of women at risk for perinatal complications and a poorer pregnancy outcome. However, a small proportion of women with nonproteinuric gestational hypertension have similar perinatal results as patients with preeclampsia11 as defined by hypertension and proteinuria. Preeclampsia is further characterized by an abnormal elevation in triglyceride levels beyond the physiologic increase of normal pregnancy,12 which is generally regarded as further evidence of the implication of lipid disturbances in its pathophysiologic mechanisms. Further studies reported that, although patients with mild gestational hypertension do not present any significant modifications in their triglyceride levels compared with controls, a subgroup of women with more severe forms of gestational hypertension have changes similar to patients with preeclampsia.13 The possible differences in the pattern of serum oxidant/antioxidant balance in pregnant
Gratacós et al. 1073
Volume 178, Number 5 Am J Obstet Gynecol
Table I. Clinical characteristics of study groups (results expressed as mean and SD) Gestational
Gestational
Mean arter-
Age (yr)
age at sampling (wk)
age at delivery (wk)
Birth weight (gm)
Proteinuria (mg/dl)
pressure (mm Hg)
27.8 (6.3) 29.7 (6.1) 28.5 (4.1) 27.7 (6.5) 30.6 (4.4)
33.3 (3.1) 36.6 (1.9) 33.5 (3.0) 33.3 (4.3) 35.0 (2.9)
38.8 (1.5) 39.0 (1.4) 34.6 (3.9) 34.9 (3.9) 38.4 (2.2)
3195 (275) 3342 (561) 2076 (926) 2146 (794) 3152 (481)
*
114 (82) 170 (55) 3085 (2594) 114 (86)
95 (8) 121 (8) 135 (9) 132 (8) 127 (9)
ial
Controls (n = 36) Mild gestational hypertension (n = 28) Severe gestational hypertension (n = 10) Preeclampsia (n = 34) Chronic hypertension (n = 20)
Table II. Lipid peroxides and vitamin E in women with different categories of hypertension (results are mean and SD) Lipid peroxide– thiobarbituric acid reactive products (nmol/ml)
Lipid peroxide– malondialdehyde (µmol/L)
Vitamin E (mmol/ml)
Corrected E (ratio vitamin E/ cholesterol)
5.5 (0.8) 5.8 (1.1) 6.8 (0.9)* 7.1 (2.0)* 5.9 (0.9)
0.39 (0.09) 0.42 (0.18) 0.47 (0.19)† 0.48 (0.18)* 0.39 (0.13)
38.1 (9.1) 43.1 (9.6) 31.3 (7.1)‡ 32.1 (3.8)† 41.3 (8.1)
14.8 (3.4) 15.4 (3.0) 12.4 (1.2)‡ 13.1 (1.9)‡ 15.1 (5.0)
Controls (n = 36) Mild gestational hypertension (n = 28) Severe gestational hypertension (n = 10) Preeclampsia (n = 34) Chronic hypertension (n = 20) Student’s t test compared with control group. *p < 0.0001. †p < 0.01. ‡p < 0.05.
women with other types of hypertension than preeclampsia remains to be established. In this study we have investigated the changes in serum lipid peroxide and vitamin E levels and placental levels of lipid peroxides in women with different forms of hypertension in pregnancy compared with nonhypertensive pregnant women. Material and methods After approval of the study protocol by the Ethical Committee of the Hospital Clinic de Barcelona, women were recruited from the Obstetrics and Gynecology Department and oral informed consent was obtained. This was a nested case-controlled study conducted within five cohorts of pregnant women. A total of 128 pregnant women were included during a 2-year period: 34 with preeclampsia, 28 with mild gestational hypertension, 10 with severe gestational hypertension, 20 with chronic hypertension, and 36 nonhypertensive pregnant women. Women in labor with ruptured membranes, multiple pregnancy, or any other concurrent medical complication were not considered for the study. The control group was a consecutive sample of pregnant women followed up at our institution undergoing a routine thirdtrimester analysis, who were delivered of a healthy term infant and without any of the exclusion criteria. The criteria for the definition of preeclampsia, gestational hypertension, and chronic hypertension were those of the World Health Organization and the International
Society for the Study of Hypertension in Pregnancy.8 In brief, gestational hypertension was diagnosed if a previously normotensive woman had two repeat (4 hours apart) diastolic blood pressure measurements of ≥90 mm Hg after the twentieth week of gestation. Preeclampsia was diagnosed if proteinuria of >300 mg/L in 24 hours was determined in a woman with gestational hypertension. Chronic hypertension was diagnosed if there was a persistent elevation in blood pressure to at least 140/90 mm Hg on two occasions before 20 weeks’ gestation. For the purposes of this study, gestational hypertension was further subclassified as severe if any of the following was present: persistent diastolic blood pressure >110 mm Hg, platelet count <105/ml, elevated aminotransferase levels, hemolysis, or neurologic involvement. All measurements in hypertensive patients were made before the administration of any antihypertensive medication. None of the participants reported the use of aspirin or vitamin E in the week before enrollment. Fasting venous blood samples were obtained from each patient and collected in Vacutainer (Becton-Dickinson Vacutainer Systems Europe) blood-collecting tubes. Tubes for vitamin E determination were protected against light exposure. Serum was separated by centrifugation, and samples were stored at –80° C until assayed. Placental tissue was immediately removed after delivery, thoroughly washed with saline solution, and immediately frozen in liquid nitrogen and stored at –80° C until analysis.
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May 1998 Am J Obstet Gynecol
Table III. Lipid peroxide–vitamin E ratio and peroxide/tocopherol ratio in women with different categories of hypertension (results are mean and SD)
Controls (n = 36) Mild gestational hypertension (n = 28) Severe gestational hypertension (n = 10) Preeclampsia (n = 34) Chronic hypertension (n = 20)
Ratio lipid peroxide– thiobarbituric acid reactive products/ corrected E
Ratio (×100) lipid peroxide– malondialdehyde/ corrected E
0.39 (0.10) 0.38 (0.09)
2.7 (0.9) 2.9 (1.5)
0.56 (0.12)*
3.8 (1.3)†
0.55 (0.19)‡ 0.43 (0.13)
3.8 (1.6)* 2.9 (1.2)
Student’s t test, compared with control group. *p < 0.01. †p < 0.05. ‡p < 0.001.
Description of techniques. Lipid peroxides were measured in serum by two methods. The first was the commonly used thiobarbituric acid method.14 A commercial kit was used (Wako Chemicals GmbH, Germany). Tetraethoxypropane was used as a standard. Results are expressed as nanograms per milliliter of thiobarbituric acid–reactive products. The second method was a modification of the thiobarbituric acid method in which the concentration of malondialdehyde, a stable end byproduct of the metabolism of lipid peroxides, is specifically measured by high-pressure liquid chromatography according to the method described by Halliwell and Chirico.15 Both techniques were also used for analysis of lipid peroxides in placental tissue. Placental tissue homogenates were prepared with 1 gm of placental tissue. Samples were homogenized with 1.15% potassium chloride buffer (tissue/buffer ratio 1:9), and lipid peroxide measurements were done after deproteinization of tissue homogenates with phosphoric acid. Results of lipid peroxides in placental tissue are expressed as lipid peroxide per milligram of protein. Protein levels in placental homogenates were determined by the pyrogallol red-molybdate method.16 Vitamin E (α-tocopherol) and tocopheryl acetate were purchased from Sigma Chemical Co. (St. Louis). Before analysis, tocopherol and tocopheryl acetate standards were adjusted for concentration by spectrophotometry, and the method was initially calibrated by means of the standard reference material SRM 968b from the National Institute for Standards and Technology (Gaithersburg, Md.). All other reagents were of analytical grade or better. Serum α-tocopherol concentration was measured by high-pressure liquid chromatography according to the method of Shearer.17 Circulating vitamin E is tightly bound to β-lipoproteins, and low concentrations of cholesterol may result in low levels of vitamin E. Serum cho-
Table IV. Placental lipid peroxide levels in women with different categories of hypertension (results are mean and SD) Thiobarbituric acid reactive products (nmol/mg protein) Controls (n = 10) Mild gestational hypertension (n = 19) Severe gestational hypertension (n = 7) Preeclampsia (n = 17) Chronic hypertension (n = 11)
Malondialdehyde (nmol/mg protein)
26.0 (8) 26.8 (9)
7.1 (0.5) 6.7 (1.2)
35.9 (18)
11.1 (3.5)
39.0 (17)* 27.7 (13)
11.3 (3.4)† 7.3 (2.3)
Student’s t test: preeclampsia versus controls. < 0.05. < 0.01.
*p †p
lesterol was also measured to calculate the ratio of vitamin E to cholesterol. Serum cholesterol levels were measured by enzymatic methods (Trinder, Bayer Diagnostics, Tarrytown, N.Y.) adapted to a Cobas Mira automated analyzer (Hoffmann-La Roche, Basel). Statistical analysis. Data were recorded into a database computer program and analyzed with the SPSS for Windows (SPSS, Chicago) statistical package. Student’s t test was used to evaluate the possible differences between the study groups. Results Table I presents the mean age, gestational age at sampling, and clinical characteristics of the study groups. As expected, women with preeclampsia and severe gestational hypertension were delivered earlier, and consequently the mean birth weight was also lower in these groups. Women with preeclampsia and severe gestational hypertension had significantly higher values of lipid peroxides as measured by thiobarbituric and high-pressure liquid chromatography methods and significantly lower values of absolute and cholesterol-corrected levels of vitamin E than healthy pregnant women (Table II). However, women with mild gestational hypertension and chronic hypertension had values similar to controls. Table III compares the mean ratios of lipid peroxides to vitamin E. All ratios were significantly increased with respect to controls in women with preeclampsia and severe gestational hypertension but showed no significant differences in the groups of mild gestational hypertension and chronic hypertension compared with healthy pregnant women. Placental tissue was available in 10 control subjects, 19 women with mild gestational hypertension, 7 with severe gestational hypertension, 17 with preeclampsia, and 11 with chronic hypertension. Again, results regarding lipid
Volume 178, Number 5 Am J Obstet Gynecol
peroxide, as measured by both techniques, were similar to those found in serum (Table IV). Lipid peroxides were significantly increased in women with preeclampsia. Women with severe gestational hypertension had similar changes, although the differences do not reach statistical significance. However, no significant differences were found for the groups of gestational hypertension and chronic hypertension. Comment To our knowledge, this is the first study evaluating the changes in lipid peroxide and vitamin E levels in women with different types of hypertension other than preeclampsia. Several studies, including this one, have reported that women with mild gestational hypertension usually have characteristics similar to pregnant women with uncomplicated chronic hypertension. Both groups appear to be older, more obese, and their perinatal results are similar to those of nonhypertensive pregnant women.18 In contrast to women with preeclampsia, women with mild gestational hypertension and uncomplicated chronic hypertension do not have elevated triglyceride levels.13 In this study serum and placental lipid peroxides in the group with mild gestational hypertension did not differ from those observed in the groups with chronic hypertension and control groups. These results further support the concept that mild gestational hypertension is a latent form of chronic hypertension in most cases.18 On the other hand, women with severe gestational hypertension appear to represent a distinct subgroup because they had serum and placental oxidative changes similar to patients with preeclampsia. The data are in line with a previous report in which patients with severe gestational hypertension showed epidemiologic and biochemical resemblance with those with preeclampsia because they had hypertriglyceridemia, were younger, had a normal body mass index, and had a poor perinatal outcome.13 This line of evidence suggests that nonproteinuric severe gestational hypertension is associated with a placental disorder, whereas milder forms of nonproteinuric hypertension are not related to placental pathologic conditions. Proteinuria is easy to detect, and it is a fairly constant sign in preeclampsia; therefore it is required for the diagnosis in widely used classifications.8, 9 However, it is accepted that proteinuria may not be found initially in a number of cases of preeclampsia or hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome, and it is not consistently found in all women with eclampsia.19 In fact, proteinuria reflects the existence of glomerular endotheliosis, but it is not a direct sign of placental involvement. Preeclampsia is extremely variable in its clinical onset and development, and there is not a constant clinical sequence of complications. Therefore, although the high susceptibility of the glomerular endothelium to
Gratacós et al. 1075
the toxic action of preeclamptic plasma makes proteinuria a fairly constant sign, its absence should not be considered an exclusion for the diagnosis of preeclampsia. Women with severe forms of gestational hypertension constitute a small proportion among all women with this condition, probably not representing more than 10% to 20% of cases. As long as a specific diagnosis of preeclampsia is not possible, it will remain difficult to distinguish true nonproteinuric pregnancy-induced hypertension from other forms of latent chronic hypertension. However, in the presence of severe hypertension or signs of severe preeclampsia (i.e., thrombocytopenia, elevated liver enzymes, or hemolysis) it is likely that these women have nonproteinuric preeclampsia. This study confirms previous results on the changes in lipid peroxide and vitamin E levels in serum and placental tissue of women with preeclampsia.3, 6, 7 We measured lipid peroxide levels by two methods, and we found a significant increase in women with preeclampsia by both. On the other hand, vitamin E was decreased, resulting in a further increase in the lipid peroxide/vitamin E ratio. Compared with nonhypertensive women, percent increase in lipid peroxide levels in women with severe gestational hypertension and preeclampsia appeared to be higher in placental tissue than in serum. 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.1 This might further indicate that serum determinations of lipid peroxide levels or consumption of vitamin E are probably indirect measurements of more profound modifications in plasma lipoproteins. On the other hand, plasma changes might be subject to more variation, whereas placental disturbances are supposed to occur more constantly. Important indirect evidence supports the notion that elevation in lipid peroxide levels is not merely a consequence of endothelial lesions. In a recent report endothelial stimulating activity of plasma from women with preeclampsia appeared to depend on the lipid fraction of plasma, and according to subsequent lipoprotein isolation the activity resided in the low-density lipoprotein (LDL) and very low-density lipoprotein fractions.20 Oxidative modification of lipoproteins appears to substantially increase their ability to adversely affect endothelial function, and oxidized LDL have been consistently shown to induce endothelial damage and dysfunction.21 On the other hand, LDL itself is heterogeneous and can be separated in subfractions on the basis of size and density,22 and smaller and more dense subfractions of LDL show a markedly increased susceptibility to oxidative modification.23 The predominance of these small dense subfractions of LDL in nonpregnant subjects is known to be associated with an increased risk of cardiovascular disease23 and is normally accompanied by hy-
1076 Gratacós et al.
pertriglyceridemia.24 It is interesting that pregnancy appears to be associated with a shift in the size and density patterns of LDL, with a predominance of smaller and more dense lipoprotein subfractions.25 It is not known whether smaller subfractions of LDL are further increased in women with preeclampsia or what is the level of oxidative modification of the different subfractions in these women. On the other hand, little is known about the possible role of other lipoproteins, such as very lowdensity lipoprotein, from which there is less evidence of its adverse actions on endothelial function or its susceptibility to oxidation. Lipoproteins might undergo intensive oxidative modification 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. Therefore elevation of lipid peroxides or consumption of vitamin E might be an indirect measure of the changes occurring in lipoproteins. Studies on the possible modifications the size and density of lipoproteins and their level of oxidative modification are now in progress. In summary, this study provides evidence supporting the notion that nonproteinuric pregnancy-induced hypertension is made up of more than one entity, one associated with a placental disorder and another in which hypertension is not related to placental pathologic conditions. This and other evidence along this line might provide useful information for future classifications of hypertensive diseases in pregnancy.
May 1998 Am J Obstet Gynecol
7.
8.
9.
10.
11. 12.
13.
14.
15.
16.
17. 18. 19. 20.
REFERENCES
1. Walsh SW. Lipid peroxidation in pregnancy. Hypertens Pregnancy 1994;13:1-32. 2. Uotila J, Tuimala R, Aarnio P, Pyykko K, Ahotupa M. Findings on lipid peroxidation and antioxidant function in hypertensive complications of pregnancy. Br J Obstet Gynaecol 1993;100:2706. 3. Wang Y, Walsh SW, Guo J, Zhang J. The imbalance between thromboxane and prostacyclin in preeclampsia is associated with an imbalance between lipid peroxides and vitamin E in maternal blood. Am J Obstet Gynecol 1991;165:1695-700. 4. Jain SK, Wise R. Relationship between elevated lipid peroxides, vitamin E deficiency, and hypertension in preeclampsia. Mol Cell Biochem 1995;151:33-8. 5. Davidge ST, Hubel CA, Brayden RD, Capelass EC, McLaughlin MK. Sera antioxidant activity in uncomplicated and preeclamptic pregnancies. Obstet Gynecol 1992;79:897-901. 6. Mikhail MS, Anyaegbuman A, Garfinkel D, Palan PR, Basu J, Romney SL. Preeclampsia and antioxidant nutrients: decreased
21. 22.
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plasma level of reduced ascorbic acid, α-tocopherol and betacarotene in women with preeclampsia. Am J Obstet Gynecol 1994;717:150-7. Wang Y, Walsh SW, Kay HH. Placental lipid peroxides and thromboxane are increased and prostacyclin is decreased in women with preeclampsia. Am J Obstet Gynecol 1992;167:946-9. Zuspan FP. The hypertensive disorders of pregnancy: report of a WHO study group. Geneva: WHO; 1987. Technical Report Series 758. Consensus Report. National high blood pressure education program working group report on high blood pressure in pregnancy. Am J Obstet Gynecol 1990;163:1689-712. Australasian Society for the Study of Hypertension in Pregnancy Consensus Statement. Management of hypertension in pregnancy: executive Summary. Med J Aust 1993;158:700-2. Brown MA, Buddie ML. The importance of nonproteinuric hypertension in pregnancy. Hypertens Pregnancy 1995;14:57-65. Potter MF, Nester PJ. The hyperlipidemia of pregnancy in normal and complicated pregnancies. Am J Obstet Gynecol 1979;133:165-70. Gratacós E, Casals E, Sanllehy C, Cararach V, Alonso PL, Fortuny A. Variation in lipid levels during pregnancy in women with different types of hypertension. Acta Obstet Gynecol Scand 1996;75:896-901. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. Halliwell H, Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993;57 Suppl:715s25s. Watanabe N, Kamei S, Ohkubo A. Urinary protein as measured with a pyrogallol red molybdate, manually, and in a Hitachi 726 automated analyzer. Clin Chem 1986;32:1551-4. Shearer MJ. Vitamins: vitamin E. In: Lim CK, editor. HPLC of small molecules. Oxford: IRL Press; 1986. p. 175-95. Perry IJ, Beevers DG. The definition of preeclampsia. Br J Obstet Gynaecol 1994;101:587-91. Redman CWG, Roberts JM. Management of pre-eclampsia. Lancet 1993;341:1451-4. Davidge ST, Signorella AP, Hubel CA, Lykins DL, Roberts JM. Distinct factors in plasma of preeclamptic women increase endothelial nitric oxide and prostacyclin. Hypertension 1996;28:758-64. Witztum JL. Susceptibility of low-density lipoprotein to oxidative modification. Am J Med 1993;94:347-9. Kuchinskiene Z, Carlson LA. Composition, concentration, and size of low density lipoproteins, and of subfractions of very low density lipoproteins from serum of normal men and women. J Lipid Res 1982;23:762-9. Chait A, Brazg RL, Tribble DL, Krauss RM. Susceptibility of small, dense, low-density lipoproteins to oxidative modification in subjects with the atherogenic lipoprotein phenotype, pattern B. Am J Med 1993;94:350-6. 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;12:128490. Silliman K, Shore V, Forte TM. Hypertriglyceridemia during late pregnancy is associated with the formation of small dense lowdensity lipoproteins and the presence of large buoyant highdensity lipoproteins. Metabolism 1994;43:1035-41.
Key words: Preeclampsia, gestational hypertension, lipid peroxides, vitamin E
Increasing evidence supports the role of abnormal lipid metabolism and circulating modified lipids in the pathophysiologic mechanisms of preeclampsia. In particular, lipid peroxides and blood oxidative imbalance are proposed as part of the cytotoxic mechanisms leading to endothelial cell injury.1 Lipid peroxides have been found to be elevated in the serum of women with preeclampsia compared with normal pregnant women,2, 3 and some correlation appears to be present between lipid peroxide levels and the severity of hypertension.4 In addition, plasma antioxidant activity5 and plasma levels of antioxidant vitamins (i.e., vitamins E and C) are low in these women.6 The concentration and production of lipid peroxides by placental tissue from women with preeclampsia is also significantly elevated. 7 However, these observations have not been evaluated in other forms of hypertension during pregnancy, such as gestational hypertension. From the Departament d’Obstetrícia i Ginecologia,a Servei de Bioquimica,b and Unitat d’Epidemiologia i Bioestadística, c Hospital Clinic de Barcelona, Universitat de Barcelona. Supported by grants from the Hospital Clínic and from the Fondo de Investigaciones Sanitarias FIS 96/1100. Received for publication July 18, 1997; revised November 14, 1997; accepted November 20, 1997. Reprint requests: Eduard Gratacós, MD, Department of Obstetrics and Gynecology, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/1/87751
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Pregnancy-induced hypertension complicates 5% to 10% of pregnancies, but the prognosis for the fetus and mother depends on the hypertensive syndrome. General agreement exists that a distinction must be made between preeclampsia and gestational hypertension, a usually more benign form of hypertension in pregnancy.8, 9 However, certain disagreement exists among different classifications as to the definition of preeclampsia, and some classifications require the presence of proteinuria,8, 9 whereas others do not.10 Indeed, the presence of proteinuria is a strong indicator of women at risk for perinatal complications and a poorer pregnancy outcome. However, a small proportion of women with nonproteinuric gestational hypertension have similar perinatal results as patients with preeclampsia11 as defined by hypertension and proteinuria. Preeclampsia is further characterized by an abnormal elevation in triglyceride levels beyond the physiologic increase of normal pregnancy,12 which is generally regarded as further evidence of the implication of lipid disturbances in its pathophysiologic mechanisms. Further studies reported that, although patients with mild gestational hypertension do not present any significant modifications in their triglyceride levels compared with controls, a subgroup of women with more severe forms of gestational hypertension have changes similar to patients with preeclampsia.13 The possible differences in the pattern of serum oxidant/antioxidant balance in pregnant
Gratacós et al. 1073
Volume 178, Number 5 Am J Obstet Gynecol
Table I. Clinical characteristics of study groups (results expressed as mean and SD) Gestational
Gestational
Mean arter-
Age (yr)
age at sampling (wk)
age at delivery (wk)
Birth weight (gm)
Proteinuria (mg/dl)
pressure (mm Hg)
27.8 (6.3) 29.7 (6.1) 28.5 (4.1) 27.7 (6.5) 30.6 (4.4)
33.3 (3.1) 36.6 (1.9) 33.5 (3.0) 33.3 (4.3) 35.0 (2.9)
38.8 (1.5) 39.0 (1.4) 34.6 (3.9) 34.9 (3.9) 38.4 (2.2)
3195 (275) 3342 (561) 2076 (926) 2146 (794) 3152 (481)
*
114 (82) 170 (55) 3085 (2594) 114 (86)
95 (8) 121 (8) 135 (9) 132 (8) 127 (9)
ial
Controls (n = 36) Mild gestational hypertension (n = 28) Severe gestational hypertension (n = 10) Preeclampsia (n = 34) Chronic hypertension (n = 20)
Table II. Lipid peroxides and vitamin E in women with different categories of hypertension (results are mean and SD) Lipid peroxide– thiobarbituric acid reactive products (nmol/ml)
Lipid peroxide– malondialdehyde (µmol/L)
Vitamin E (mmol/ml)
Corrected E (ratio vitamin E/ cholesterol)
5.5 (0.8) 5.8 (1.1) 6.8 (0.9)* 7.1 (2.0)* 5.9 (0.9)
0.39 (0.09) 0.42 (0.18) 0.47 (0.19)† 0.48 (0.18)* 0.39 (0.13)
38.1 (9.1) 43.1 (9.6) 31.3 (7.1)‡ 32.1 (3.8)† 41.3 (8.1)
14.8 (3.4) 15.4 (3.0) 12.4 (1.2)‡ 13.1 (1.9)‡ 15.1 (5.0)
Controls (n = 36) Mild gestational hypertension (n = 28) Severe gestational hypertension (n = 10) Preeclampsia (n = 34) Chronic hypertension (n = 20) Student’s t test compared with control group. *p < 0.0001. †p < 0.01. ‡p < 0.05.
women with other types of hypertension than preeclampsia remains to be established. In this study we have investigated the changes in serum lipid peroxide and vitamin E levels and placental levels of lipid peroxides in women with different forms of hypertension in pregnancy compared with nonhypertensive pregnant women. Material and methods After approval of the study protocol by the Ethical Committee of the Hospital Clinic de Barcelona, women were recruited from the Obstetrics and Gynecology Department and oral informed consent was obtained. This was a nested case-controlled study conducted within five cohorts of pregnant women. A total of 128 pregnant women were included during a 2-year period: 34 with preeclampsia, 28 with mild gestational hypertension, 10 with severe gestational hypertension, 20 with chronic hypertension, and 36 nonhypertensive pregnant women. Women in labor with ruptured membranes, multiple pregnancy, or any other concurrent medical complication were not considered for the study. The control group was a consecutive sample of pregnant women followed up at our institution undergoing a routine thirdtrimester analysis, who were delivered of a healthy term infant and without any of the exclusion criteria. The criteria for the definition of preeclampsia, gestational hypertension, and chronic hypertension were those of the World Health Organization and the International
Society for the Study of Hypertension in Pregnancy.8 In brief, gestational hypertension was diagnosed if a previously normotensive woman had two repeat (4 hours apart) diastolic blood pressure measurements of ≥90 mm Hg after the twentieth week of gestation. Preeclampsia was diagnosed if proteinuria of >300 mg/L in 24 hours was determined in a woman with gestational hypertension. Chronic hypertension was diagnosed if there was a persistent elevation in blood pressure to at least 140/90 mm Hg on two occasions before 20 weeks’ gestation. For the purposes of this study, gestational hypertension was further subclassified as severe if any of the following was present: persistent diastolic blood pressure >110 mm Hg, platelet count <105/ml, elevated aminotransferase levels, hemolysis, or neurologic involvement. All measurements in hypertensive patients were made before the administration of any antihypertensive medication. None of the participants reported the use of aspirin or vitamin E in the week before enrollment. Fasting venous blood samples were obtained from each patient and collected in Vacutainer (Becton-Dickinson Vacutainer Systems Europe) blood-collecting tubes. Tubes for vitamin E determination were protected against light exposure. Serum was separated by centrifugation, and samples were stored at –80° C until assayed. Placental tissue was immediately removed after delivery, thoroughly washed with saline solution, and immediately frozen in liquid nitrogen and stored at –80° C until analysis.
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May 1998 Am J Obstet Gynecol
Table III. Lipid peroxide–vitamin E ratio and peroxide/tocopherol ratio in women with different categories of hypertension (results are mean and SD)
Controls (n = 36) Mild gestational hypertension (n = 28) Severe gestational hypertension (n = 10) Preeclampsia (n = 34) Chronic hypertension (n = 20)
Ratio lipid peroxide– thiobarbituric acid reactive products/ corrected E
Ratio (×100) lipid peroxide– malondialdehyde/ corrected E
0.39 (0.10) 0.38 (0.09)
2.7 (0.9) 2.9 (1.5)
0.56 (0.12)*
3.8 (1.3)†
0.55 (0.19)‡ 0.43 (0.13)
3.8 (1.6)* 2.9 (1.2)
Student’s t test, compared with control group. *p < 0.01. †p < 0.05. ‡p < 0.001.
Description of techniques. Lipid peroxides were measured in serum by two methods. The first was the commonly used thiobarbituric acid method.14 A commercial kit was used (Wako Chemicals GmbH, Germany). Tetraethoxypropane was used as a standard. Results are expressed as nanograms per milliliter of thiobarbituric acid–reactive products. The second method was a modification of the thiobarbituric acid method in which the concentration of malondialdehyde, a stable end byproduct of the metabolism of lipid peroxides, is specifically measured by high-pressure liquid chromatography according to the method described by Halliwell and Chirico.15 Both techniques were also used for analysis of lipid peroxides in placental tissue. Placental tissue homogenates were prepared with 1 gm of placental tissue. Samples were homogenized with 1.15% potassium chloride buffer (tissue/buffer ratio 1:9), and lipid peroxide measurements were done after deproteinization of tissue homogenates with phosphoric acid. Results of lipid peroxides in placental tissue are expressed as lipid peroxide per milligram of protein. Protein levels in placental homogenates were determined by the pyrogallol red-molybdate method.16 Vitamin E (α-tocopherol) and tocopheryl acetate were purchased from Sigma Chemical Co. (St. Louis). Before analysis, tocopherol and tocopheryl acetate standards were adjusted for concentration by spectrophotometry, and the method was initially calibrated by means of the standard reference material SRM 968b from the National Institute for Standards and Technology (Gaithersburg, Md.). All other reagents were of analytical grade or better. Serum α-tocopherol concentration was measured by high-pressure liquid chromatography according to the method of Shearer.17 Circulating vitamin E is tightly bound to β-lipoproteins, and low concentrations of cholesterol may result in low levels of vitamin E. Serum cho-
Table IV. Placental lipid peroxide levels in women with different categories of hypertension (results are mean and SD) Thiobarbituric acid reactive products (nmol/mg protein) Controls (n = 10) Mild gestational hypertension (n = 19) Severe gestational hypertension (n = 7) Preeclampsia (n = 17) Chronic hypertension (n = 11)
Malondialdehyde (nmol/mg protein)
26.0 (8) 26.8 (9)
7.1 (0.5) 6.7 (1.2)
35.9 (18)
11.1 (3.5)
39.0 (17)* 27.7 (13)
11.3 (3.4)† 7.3 (2.3)
Student’s t test: preeclampsia versus controls. < 0.05. < 0.01.
*p †p
lesterol was also measured to calculate the ratio of vitamin E to cholesterol. Serum cholesterol levels were measured by enzymatic methods (Trinder, Bayer Diagnostics, Tarrytown, N.Y.) adapted to a Cobas Mira automated analyzer (Hoffmann-La Roche, Basel). Statistical analysis. Data were recorded into a database computer program and analyzed with the SPSS for Windows (SPSS, Chicago) statistical package. Student’s t test was used to evaluate the possible differences between the study groups. Results Table I presents the mean age, gestational age at sampling, and clinical characteristics of the study groups. As expected, women with preeclampsia and severe gestational hypertension were delivered earlier, and consequently the mean birth weight was also lower in these groups. Women with preeclampsia and severe gestational hypertension had significantly higher values of lipid peroxides as measured by thiobarbituric and high-pressure liquid chromatography methods and significantly lower values of absolute and cholesterol-corrected levels of vitamin E than healthy pregnant women (Table II). However, women with mild gestational hypertension and chronic hypertension had values similar to controls. Table III compares the mean ratios of lipid peroxides to vitamin E. All ratios were significantly increased with respect to controls in women with preeclampsia and severe gestational hypertension but showed no significant differences in the groups of mild gestational hypertension and chronic hypertension compared with healthy pregnant women. Placental tissue was available in 10 control subjects, 19 women with mild gestational hypertension, 7 with severe gestational hypertension, 17 with preeclampsia, and 11 with chronic hypertension. Again, results regarding lipid
Volume 178, Number 5 Am J Obstet Gynecol
peroxide, as measured by both techniques, were similar to those found in serum (Table IV). Lipid peroxides were significantly increased in women with preeclampsia. Women with severe gestational hypertension had similar changes, although the differences do not reach statistical significance. However, no significant differences were found for the groups of gestational hypertension and chronic hypertension. Comment To our knowledge, this is the first study evaluating the changes in lipid peroxide and vitamin E levels in women with different types of hypertension other than preeclampsia. Several studies, including this one, have reported that women with mild gestational hypertension usually have characteristics similar to pregnant women with uncomplicated chronic hypertension. Both groups appear to be older, more obese, and their perinatal results are similar to those of nonhypertensive pregnant women.18 In contrast to women with preeclampsia, women with mild gestational hypertension and uncomplicated chronic hypertension do not have elevated triglyceride levels.13 In this study serum and placental lipid peroxides in the group with mild gestational hypertension did not differ from those observed in the groups with chronic hypertension and control groups. These results further support the concept that mild gestational hypertension is a latent form of chronic hypertension in most cases.18 On the other hand, women with severe gestational hypertension appear to represent a distinct subgroup because they had serum and placental oxidative changes similar to patients with preeclampsia. The data are in line with a previous report in which patients with severe gestational hypertension showed epidemiologic and biochemical resemblance with those with preeclampsia because they had hypertriglyceridemia, were younger, had a normal body mass index, and had a poor perinatal outcome.13 This line of evidence suggests that nonproteinuric severe gestational hypertension is associated with a placental disorder, whereas milder forms of nonproteinuric hypertension are not related to placental pathologic conditions. Proteinuria is easy to detect, and it is a fairly constant sign in preeclampsia; therefore it is required for the diagnosis in widely used classifications.8, 9 However, it is accepted that proteinuria may not be found initially in a number of cases of preeclampsia or hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome, and it is not consistently found in all women with eclampsia.19 In fact, proteinuria reflects the existence of glomerular endotheliosis, but it is not a direct sign of placental involvement. Preeclampsia is extremely variable in its clinical onset and development, and there is not a constant clinical sequence of complications. Therefore, although the high susceptibility of the glomerular endothelium to
Gratacós et al. 1075
the toxic action of preeclamptic plasma makes proteinuria a fairly constant sign, its absence should not be considered an exclusion for the diagnosis of preeclampsia. Women with severe forms of gestational hypertension constitute a small proportion among all women with this condition, probably not representing more than 10% to 20% of cases. As long as a specific diagnosis of preeclampsia is not possible, it will remain difficult to distinguish true nonproteinuric pregnancy-induced hypertension from other forms of latent chronic hypertension. However, in the presence of severe hypertension or signs of severe preeclampsia (i.e., thrombocytopenia, elevated liver enzymes, or hemolysis) it is likely that these women have nonproteinuric preeclampsia. This study confirms previous results on the changes in lipid peroxide and vitamin E levels in serum and placental tissue of women with preeclampsia.3, 6, 7 We measured lipid peroxide levels by two methods, and we found a significant increase in women with preeclampsia by both. On the other hand, vitamin E was decreased, resulting in a further increase in the lipid peroxide/vitamin E ratio. Compared with nonhypertensive women, percent increase in lipid peroxide levels in women with severe gestational hypertension and preeclampsia appeared to be higher in placental tissue than in serum. 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.1 This might further indicate that serum determinations of lipid peroxide levels or consumption of vitamin E are probably indirect measurements of more profound modifications in plasma lipoproteins. On the other hand, plasma changes might be subject to more variation, whereas placental disturbances are supposed to occur more constantly. Important indirect evidence supports the notion that elevation in lipid peroxide levels is not merely a consequence of endothelial lesions. In a recent report endothelial stimulating activity of plasma from women with preeclampsia appeared to depend on the lipid fraction of plasma, and according to subsequent lipoprotein isolation the activity resided in the low-density lipoprotein (LDL) and very low-density lipoprotein fractions.20 Oxidative modification of lipoproteins appears to substantially increase their ability to adversely affect endothelial function, and oxidized LDL have been consistently shown to induce endothelial damage and dysfunction.21 On the other hand, LDL itself is heterogeneous and can be separated in subfractions on the basis of size and density,22 and smaller and more dense subfractions of LDL show a markedly increased susceptibility to oxidative modification.23 The predominance of these small dense subfractions of LDL in nonpregnant subjects is known to be associated with an increased risk of cardiovascular disease23 and is normally accompanied by hy-
1076 Gratacós et al.
pertriglyceridemia.24 It is interesting that pregnancy appears to be associated with a shift in the size and density patterns of LDL, with a predominance of smaller and more dense lipoprotein subfractions.25 It is not known whether smaller subfractions of LDL are further increased in women with preeclampsia or what is the level of oxidative modification of the different subfractions in these women. On the other hand, little is known about the possible role of other lipoproteins, such as very lowdensity lipoprotein, from which there is less evidence of its adverse actions on endothelial function or its susceptibility to oxidation. Lipoproteins might undergo intensive oxidative modification 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. Therefore elevation of lipid peroxides or consumption of vitamin E might be an indirect measure of the changes occurring in lipoproteins. Studies on the possible modifications the size and density of lipoproteins and their level of oxidative modification are now in progress. In summary, this study provides evidence supporting the notion that nonproteinuric pregnancy-induced hypertension is made up of more than one entity, one associated with a placental disorder and another in which hypertension is not related to placental pathologic conditions. This and other evidence along this line might provide useful information for future classifications of hypertensive diseases in pregnancy.
May 1998 Am J Obstet Gynecol
7.
8.
9.
10.
11. 12.
13.
14.
15.
16.
17. 18. 19. 20.
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