1 Department of Epidemiology, University of Washington, Seattle, WA.
2 Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA.
3 Center for Perinatal Studies, Swedish Medical Center, Seattle, WA.
4 Dos de Mayo Hospital, Lima, Peru.
5 Materno-Perinatal Institute, Lima, Peru.
6 Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA.
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ABSTRACT |
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carotenoids; pre-eclampsia; pregnancy; risk factors; vitamin A; vitamin E
Abbreviations: CI, confidence interval; HPLC, high performance liquid chromatography; OR, odds ratio; SE, standard error
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INTRODUCTION |
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To our knowledge, no results regarding the magnitude of the odds ratio of preeclampsia associated with varying concentrations of carotenoids, tocopherols, and retinol have been reported. Additionally, potential confounding factors of preeclampsia were not evaluated in most previous studies. We studied a large group of Peruvian women to examine the relation of plasma concentrations of carotenoids, tocopherols, and retinol, measured in the third trimester of pregnancy, with the risk of preeclampsia.
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MATERIALS AND METHODS |
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A standardized, structured questionnaire was used to collect information regarding maternal sociodemographic, medical, reproductive, and lifestyle characteristics during in-person interviews. All interviews were conducted in the hospital. Maternal and infant records were reviewed to collect detailed information concerning antepartum, labor, and delivery characteristics and the condition of the newborn infants. Maternal anthropometric measures (height, weight, and midarm circumference) were taken during the participants' hospital stay. Gestational age was based on the date of the last menstrual period and was confirmed by ultrasound examination. Prepregnancy body mass index, used as a measure of overall maternal adiposity, was calculated as weight (kg)/height (m)2. Blood samples collected in 10-ml ethylenediaminetetraacetic acid (EDTA) Vacutainer tubes (Becton Dickinson and Company, E. Rutherford, New Jersey) were immediately transported in a cooler with ice to the Blood Bank Laboratory of Dos de Mayo Hospital. Upon arrival at the laboratory, blood was centrifuged and plasma was divided into 1.0- to 2.0-ml aliquots, placed in cryovials, and stored at -70°C. Specimens were shipped on dry ice with blue ice packs to the United States for biochemical analyses.
Blood samples were available for 180 cases and 196 control subjects. After the exclusion of six cases and three control subjects with chronic hypertension diagnosed prior to pregnancy or during the first 20 weeks of the index pregnancy, 174 preeclampsia cases and 193 normotensive control subjects remained for study. Because maternal plasma antioxidant nutrients and lipid concentrations may change rapidly after parturition (19), we further excluded 49 preeclampsia cases and 14 controls for whom blood samples were drawn after delivery, thus leaving 125 cases and 179 controls for this research. Maternal sociodemographic and clinical characteristics were not materially different for those excluded subjects compared with those retained for this analysis.
Plasma antioxidant nutrients were measured by high performance liquid chromatography (HPLC). The HPLC assay was modified to expand analysis to include determination of lutein, zeaxanthin, and ß-cryptoxanthin concentrations. The extraction of analytes from plasma, the quality control parameters, and the HPLC methods were previously published (20). Briefly, a hexane extract of plasma was injected onto a 3-mm C-18 Spherisorb ODS-2 HPLC column (Alltech, Deerfield, Illinois) and eluted with an isocratic solvent consisting of 73 percent acetonitrile, 12 percent tetrahydrofuran, 8 percent methanol, 7 percent water, 0.025 percent ammonium acetate, and 0.05 percent diethylamine (volume/volume) at a flow rate of 1.2 ml/minute. Lutein, zeaxanthin, ß-cryptoxanthin, and lycopene were detected at 476 nm,
-carotene and ß-carotene were detected at 452 nm,
-tocopherol and
-tocopherol were detected at 292 nm, and retinol was detected at 325 nm. The coefficient of variation for pooled quality control samples was
10 percent for all analytes. Plasma cholesterol was measured to calculate the ratio of vitamin E to cholesterol. Plasma total cholesterol concentrations were measured enzymatically (18
). Total cholesterol measurements were standardized by the Lipid Standardization Program of the Centers for Disease Control and Prevention, Atlanta, Georgia. Triglycerides were analyzed by a glycerol phosphate oxidation assay. All laboratory analyses were performed without knowledge of pregnancy outcome.
The plasma tocopherol concentration is influenced by plasma lipoproteins that are transport molecules of the antioxidant (21). We controlled for potential confounding by plasma lipids in three ways. First, we calculated the ratio of plasma
-tocopherol to plasma total cholesterol concentrations. The ratio of
-tocopherol to cholesterol is considered by some investigators to be a biologically more relevant marker of vitamin E status than is
-tocopherol alone (22
25
). Second, as described by Thurnham et al. (22
), we calculated plasma total lipids as (2 x cholesterol + triglycerides) and calculated the ratio of
-tocopherol to total lipids. Third, we adjusted for plasma concentrations of cholesterol and/or plasma total lipids when analyzing the relation of plasma antioxidants with the risk of preeclampsia.
We examined the frequency distributions of maternal sociodemographic characteristics and medical and reproductive histories according to case-control status. Comparisons of categorical variables were made between case and control subjects using chi-squared or Fisher's exact tests. In order to compare our results with those from previous clinical studies concerning the relation between plasma antioxidants and preeclampsia, we calculated the mean and median values of plasma antioxidant concentrations for the two study groups. Student's t tests were used to evaluate mean differences in maternal plasma concentrations of antioxidant nutrients between cases and controls. Furthermore, because of the extremely skewed distributions of plasma concentrations of ß-cryptoxanthin, the Mann-Whitney U test was used to assess median differences for that analyte.
To further evaluate the association between preeclampsia and plasma antioxidants, we categorized each subject according to quartiles determined by the distribution of antioxidant concentrations in controls. Using the lowest category as the referent group, we calculated odds ratios (i.e., estimates of relative risk) and their 95 percent confidence intervals. The Mantel extension test for linear trend in proportions (26) was used in univariate analyses to test for a linear component of trend in the risk of preeclampsia in relation to each antioxidant. Logistic regression procedures were used to calculate maximum likelihood estimates for the coefficients and their standard errors. These estimates and their standard errors were then used to calculate odds ratios and 95 percent confidence intervals, adjusted for confounders (27
). In logistic regression models, the significance for monotonic trend was assessed by treating the four quartiles as a continuous variable after assigning a score to each quartile (27
). To assess confounding, we entered variables into a logistic regression model one at a time and then compared the adjusted and unadjusted odds ratios (27
). Final logistic regression models included covariates that altered unadjusted odds ratios by at least 10 percent, as well as those covariates with a priori interest (e.g., maternal age and parity). The candidate confounding variables considered were maternal age, race/ethnicity, educational attainment, smoking during pregnancy, use of prenatal vitamins, plasma cholesterol concentrations, marital status, parity, prepregnancy body mass index, gestational age at blood collection, and whether the pregnancy was planned. All reported p values are two tailed, and confidence intervals were calculated at the 95 percent level.
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RESULTS |
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DISCUSSION |
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Epidemiologic studies relating plasma concentrations of lipid-soluble antioxidants or prooxidants to preeclampsia risk are limited. Furthermore, inferences from the few available prior studies have been limited by their relatively small sample size and their inconsistent findings. Results from previous studies of maternal plasma carotenoids in preeclamptic and normotensive pregnant women, for instance, have been inconsistent (9, 28
, 29
). Our results are in agreement with those of Jendryczko and Dro
d
(29
), who reported that plasma concentrations of
-carotene, ß-carotene, lutein, and lycopene were similar for preeclamptic women and normotensive pregnant women. Our results, however, did not corroborate the findings reported by Ziari et al. (28
), who noted that the mean plasma ß-carotene concentrations among preeclamptic women were 40 percent lower as compared with the mean concentrations in normotensive pregnant West African women.
Relatively few investigators have assessed the extent to which maternal retinol concentrations are altered in preeclamptic versus normotensive pregnancies. Our finding of decreased retinol concentrations in preeclamptic pregnancies is consistent with results from previous studies (28, 29
). Ziari et al. (28
) noted that mean maternal plasma retinol concentrations were 37 percent lower in the nine preeclampsia cases studied as compared with 16 normotensive controls (15.3 mg/dl vs. 24.2 mg/dl; p < 0.01).
Results from previous studies have suggested that alterations in maternal plasma or serum -tocopherol concentrations are associated with preeclampsia. Several investigators have noted that women with preeclampsia, as compared with normotensive pregnant women, have lower
-tocopherol concentrations (9
, 28
, 30
, 31
). Other investigators (13
, 15
), however, have reported that preeclamptics have higher mean
-tocopherol concentrations as compared with normotensive controls. In a study of women delivering in the Tampere University Hospital, Finland, Uotila et al. (13
) reported that women with severe preeclampsia had
-tocopherol concentrations that were 25 percent higher than concentrations measured in normotensive pregnant women (mean plasma concentrations of 39.0 (standard error (SE), 8.6) vs. 31.2 (SE, 5.9) µmol/liter; mean gestational age at sampling of 34.3 (SE, 2.2) vs. 35.3 (SE, 1.5) weeks). This finding of elevated
-tocopherol among preeclamptic women as compared with normotensive pregnant women was corroborated by Schiff et al. (15
). In their study of US women, the authors noted that mean maternal plasma
-tocopherol concentrations were 20 percent higher in preeclampsia cases as compared with controls (1.43 (SE, 0.46) vs. 1.15 (SE, 0.32) mg/dl). In accordance with the results reported by Uotila et al. (13
) and Schiff et al. (15
), we noted that mean plasma
-tocopherol concentrations were 11 percent higher in cases as compared with controls. Moreover, we observed a statistically significant linear trend in the risk of preeclampsia across increasing quartiles of
-tocopherol concentrations.
Differences in study design, limited statistical power, differences in population characteristics (such as maternal age, race/ethnicity, and country of residence), failure to adjust for gestational age at blood collection, and failure to adjust lipophilic antioxidants for maternal lipid status may all have contributed to inconsistencies in results from previous studies. Variations in gestational age-specific plasma volume expansion and hyperlipidemia, for instance, have generally not been accounted for in most previous studies. We adjusted for gestational age at blood collection as a means to control for confounding by maternal plasma volume expansion. Furthermore, we controlled for potential confounding by maternal hyperlipidemia in three ways. We adjusted for plasma total cholesterol or total plasma lipids when analyzing the relation of plasma antioxidants with the risk of preeclampsia. We also calculated the ratio of plasma -tocopherol to plasma total cholesterol and the ratio of plasma
-tocopherol to plasma total lipids as suggested by some previous investigators (21
, 22
, 24
).
Potential limitations and strengths of our research must be considered when interpreting our results. First, because maternal blood samples were collected when preeclampsia became clinically evident, we cannot determine whether the observed associations between maternal plasma concentrations of antioxidants may be attributable to disease-related alterations in maternal antioxidant metabolism or whether the alterations in plasma antioxidant concentrations are causally related to preeclampsia. Second, lack of information pertaining to maternal dietary habits limited our ability to assess maternal dietary intake of these antioxidants and the risk of preeclampsia. Moreover, we were not able to measure the water-soluble antioxidant, ascorbic acid, and antioxidant enzymes including superoxide dismutase and glutathione peroxidase. Ascorbic acid and antioxidant enzymes, as well as the lipid-soluble antioxidants we studied, are all important in the defense against oxidative stress (9). Ascorbic acid, for instance, is known to function as the first-line antioxidant defense against free oxygen radicals present primarily in plasma (32
).
-Tocopherol and ascorbic acid have been shown to act syngeristically (33
, 34
) in preventing lipid peroxidation in vitro. Specifically, results from quantitative studies designed to directly measure antioxidant consumption and free radical generation under controlled systems have confirmed the antioxidant activities of
-tocopherol in early stages of low density lipoprotein oxidation (35
). However, under identical experimental conditions
-tocopherol, in the absence of ascorbic acid, has been shown to act as a chain-transfer agent (prooxidant). These observations suggest that our inability to measure ascorbic acid limited our capacity to fully explore the relation between maternal antioxidant status and preeclampsia.
The relatively large sample size of our study allowed us to assess relative risk estimates for preeclampsia with varying concentrations of antioxidants while adjusting for potential confounders. Nonetheless, we cannot exclude the possibility that residual confounding may still have affected the reported odds ratios. Differential misclassification of maternal plasma antioxidant concentrations is unlikely, as all laboratory analyses were conducted without knowledge of participants' pregnancy outcome.
Our results together with those from previous studies (1, 2
, 9
, 10
, 13
, 15
, 28
, 30
, 31
) suggest a paradoxic relation between maternal plasma antioxidants and preeclampsia risk. The pathophysiologic mechanisms for the observed association are not yet fully understood. However, several lines of evidence suggest that possible biologic mechanisms for the association between elevated
-tocopherol and increased peroxidation in preeclampsia are complex. As noted earlier, results from in vitro studies suggested that, in the absence of aqueous antioxidants, such as ascorbic acid,
-tocopherol can act as a prooxidant (36
40
). It is possible that, under the physiologic oxidative stress commonly identified in preeclampsia, after first-line defense antioxidants such as ascorbate acid are consumed, high concentrations of
-tocopherol may act as a prooxidant rather than as an antioxidant. The increased plasma concentrations of
-tocopherol in preeclamptic women may also be a compensatory increase in response to the elevated oxidative stress of preeclampsia, as suggested by a previous study (13
).
Moreover, elevated -tocopherol in preeclamptic, compared with normotensive, women may, in part, be attributed to altered placental physiology. Extensive changes in the placenta including thickening of the trophoblastic basement membrane, placental infarction, proliferation of the cytotrophoblast, and retroplacental hematoma have been observed in preeclamptic women more frequently than in normotensive pregnant women (41
). It is likely that preeclampsia-related placental abnormalities may result in a decrease in the transfer of some nutrients, including
-tocopherol, to the fetus. As a result, more
-tocopherol will be retained in the maternal circulation in preeclamptic women compared with normotensive pregnant women. Additional studies of the placental transfer of antioxidant vitamins in preeclamptic and normotensive pregnancy are needed.
In summary, we have shown that plasma retinol concentrations were negatively associated with an increased risk of preeclampsia. Plasma -tocopherol concentrations, however, were positively associated with an increased risk of preeclampsia. This association persisted after controlling for maternal plasma lipid status and other potential confounding factors. Although the biologic mechanisms for this statistical association are presently not well understood, our results, when taken together with those of others, suggest that preeclampsia may not be a state of global antioxidant deficiency in maternal peripheral circulation. Future prospective longitudinal studies involving measurements of concentrations of antioxidant nutrients and enzymes in blood and placental tissue are needed to confirm and expand upon our findings. Information about maternal dietary intake and use of dietary supplements of antioxidants should also be collected in future studies. At present, the results from our study and those of others (1
, 2
, 9
, 10
, 13
, 15
, 28
, 30
, 31
) suggest that the role of
-tocopherol in the pathogenesis of preeclampsia may be complex.
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ACKNOWLEDGMENTS |
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The authors thank Mirtha Grande, Elena Sanchez, Nelly Toledo, Hong Tang, Mohammed Adem, and June Hu for their skillful technical assistance.
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NOTES |
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REFERENCES |
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