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Department of Obstetrics and Gynecology (X.C., T.O.S.) and Surgery (M.J.L., M.R.D., T.P.S.), University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084
Address all correspondence and requests for reprints to: Dr. Theresa O. Scholl, Department of Obstetrics and Gynecology, University of Medicine and Dentistry of New Jersey–School of Osteopathic Medicine, Two Medical Drive, Science Center, Suite 185, Stratford, New Jersey 08084. E-mail: scholl{at}umdnj.edu.
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Abstract |
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Introduction |
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During normal pregnancy there is a physiological increase in insulin resistance that is paralleled by increasing oxidative stress and alterations in antioxidant status (1, 10). Data on changes of GPx activity during pregnancy are conflicting (11, 12, 13). There is virtually no information on the relationship between insulin or insulin resistance and GPx activity in healthy pregnant women; thus, whether these parallel changes are linked is unknown. Although there is some evidence that FFAs, especially long-chain polyunsaturated fatty acids (PUFAs) play a role in modulation of GPx activity (7, 8, 9), there are no data on human normal pregnancy. The contribution of maternal factors such as the mother’s ethnic background to GPx activity is unstudied as well. We therefore examined GPx activity, indicators of insulin resistance, and dietary fat intake prospectively among 408 normotensive nondiabetic gravidas from three ethnic groups (African American, Hispanic, Caucasian) in Camden, NJ.
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Subjects and Methods |
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Data were collected as part of the Camden Study, a prospective cohort study of maternal nutrition and pregnancy outcome in young, generally healthy women residing in one of the poorest cities in the continental United States (14, 15). The institutional review board at the University of Medicine and Dentistry of New Jersey–School of Osteopathic Medicine approved the study protocol. Informed written consent was obtained from each participant after explanation of the nature and purpose of the study.
Subjects
Study participants were enrolled between 1998 and 2002 for prenatal care. Less than 5% of the women screened for eligibility were excluded from participation because of serious nonobstetric problem (e.g. Lupus, type 1 or 2 diabetes, seizure disorders, malignancies, drug or alcohol abuse, and psychiatric problems). Eighty percent of the patients who were eligible agreed to participate in this study. The final total of 408 pregnant women (166 African Americans, 186 Hispanics, and 56 Caucasians) were included in this analysis after the exclusions of 25 women who developed gestational diabetes mellitus in the course of pregnancy, 53 who developed pregnancy-induced hypertension, and/or preeclampsia, and four Asian women (inadequate statistical power).
Socioeconomic, demographic, and lifestyle data were obtained by interview at entry to care, and updated at wk 20 and 28 of gestation. A 24-h recall of the previous day’s diet was obtained on the same schedule, processed with database from the Campbell Institute of Research and Technology (16, 17), which was updated from the United States Department of Agriculture’s most current data sets, the Nutrient Database for Standard Reference (release 13, 2000) and the Survey Nutrient Database for the Continuing Survey of Food Intakes by Individuals (1994–1996). Total daily intake of energy, fat, PUFAs, n-3 fatty acids (n-3 FA) (sum of 18:3, 22:6, 20:5, and 22:5) and n-6 fatty acids (n-6 FA) (sum of 18:2 and 20:4) were calculated for entry values and three 24-h recalls were averaged for the values of pregnancy. Ethnicity was self-defined. Maternal weight was measured at each visit; height was measured at entry to prenatal care, and pregravid weight was obtained by recall. Body mass index (BMI) was computed as pregravid weight for height (kilograms per square meter). Information on current and past pregnancy outcomes, complications, and infant abnormalities was abstracted from the prenatal records, the delivery record, delivery logbooks, and the infant’s chart. Gestation duration was based on gestation from the gravidas’ last normal menstrual period confirmed or modified by ultrasound.
Samples collection and analytic procedures
Fasting (
8 h) blood samples were collected at entry to care (entry, 16.01 ± 0.30 of gestation weeks) and during the third trimester (29.89 ± 0.16 of gestation weeks) from study participants. Blood samples collected at each visit were immediately refrigerated and centrifuged at 4 C. Plasma and serum samples were stored at -70 C until assayed. Heparinized whole blood was centrifuged and the plasma was aspirated. The erythrocyte sediment was washed and stored at -70 C until assayed for GPx.
GPx activity was determined spectrophotometrically from a lysate of the washed packed erythrocytes fraction by using a test reagent kit (Calbiochem-Novabiochem Corp., San Diego, CA). Absorbance was measured at 340 nm. Results are expressed as milliunits of GPx activity per milligram of hemoglobin (Hb). Hb was assayed by the commercial cyanmethemoglobin method (Sigma Diagnostics, St. Louis, MO). Fasting plasma glucose was measured by the glucose oxidase method (Sigma Diagnostics). Serum insulin was determined by RIA using a kit with a specific antibody that cross-reacts only minimally (<0.2%) with proinsulin and has a high sensitivity (2 µU/ml or 12 pmol/liter) (Linco, St. Charles, MO). Plasma C-peptide was determined using a RIA kit that has a high sensitivity (0.1 ng/ml or 0.033 nmol/liter) and low cross-activity to proinsulin (<4%) (Linco). The coefficient of variation within and between assays was 3.2% and 6.3% for C-peptide, 3.5% and 6.5% for insulin, 1.5% and 3.0% for glucose, and 2.0% and 5.4% for GPx activity.
Calculation of homeostasis model of assessment for insulin resistance (HOMA IR)
It has been suggested that the HOMA IR obtained from the same day’s fasting specimens of glucose and insulin provides reasonable estimates of insulin resistance or sensitivity, especially in the large-scale epidemiological studies (18, 19). The use of HOMA IR has been validated in studies of pregnant women with normal glucose tolerance as well as in gravidas with gestational diabetes mellitus (20). HOMA IR is computed as the product of fasting plasma glucose and fasting serum insulin divided by a constant (22.5), under the assumption that young normal subjects have an insulin resistance of 1.0 (18, 20).
Statistical analyses
ANOVA was used to generate means for GPx activity, metabolic measurements, and dietary fat intake after adjusting for potential confounding variables (age, pregravid BMI, cigarette smoking, parity, and ethnicity). Confounding was assessed by comparing crude and adjusted regression coefficients. Results from these models were expressed as means ± SEM. The least squares means from the models were tested among ethnic groups and sampling points using Bonferroni’s correction for multiple comparisons. Partial correlation analysis (adjusted for total energy intake) was performed to determine the relation between dietary fat intake and GPx activity.
The multiple linear mixed models were used to investigate the influence of metabolic parameters on GPx activity and the pattern of GPx change during pregnancy. The analysis assumes that errors are correlated within an individual, observations are independent across individuals, and all missing observations are missing at random (21). GPx activity was used as the dependent variable. Visit (coded as entry and third trimester) was used as a class variable; age, pregravid BMI, cigarette smoking, parity, and ethnicity were used as fixed effects, and subject(s) was the random effect. Separate models were used to estimate the influence of each of the metabolic variables (insulin, glucose, C-peptide, and HOMA IR) on GPx activity. All statistical procedures were performed using SAS version 8.0 (SAS Institute, Inc., Cary, NC).
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Results |
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. African-American, Hispanic, and Caucasian women were comparable in age, parity, and pregravid BMI, but Caucasians were about twice as likely to smoke as other gravidas. For the entire sample of women, mean age at entry was 21.86 ± 0.23 yr (mean ± SE) with a mean pregravid BMI of 25.34 ± 0.29 kg/m2.
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Fasting GPx, insulin, glucose, C-peptide, and HOMA IR levels are presented in Table 2. In comparison with entry data, GPx activity, insulin and C-peptide concentrations, and HOMA IR levels all had increased significantly by the third trimester (P < 0.0001 for each). Fasting plasma glucose concentrations also increased by the third trimester, and although the difference was small, it was statistically significant (P < 0.03).
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Separate models were fit for each of the metabolic parameters, insulin, glucose, C-peptide, and HOMA IR, that also included the base model. Estimated regression coefficients for each model are presented in Table 3. The relation between GPx activity and the variables included in the base model remained unchanged after adding the metabolic parameter. In each instance there was a significant and positive effect of the metabolic variable on GPX activity. These included fasting insulin (ß = 0.009 mU/mg Hb·pmol per liter of insulin, P = 0.001), glucose (ß = 0.975 mU/mg Hb·mmol/liter of glucose, P = 0.037), C-peptide (ß = 1.537 mU/mg Hb·nmol per liter of C-peptide, P = 0.003), and HOMA IR (ß = 0.209 mU/mg Hb per HOMA IR, P = 0.002). The magnitude of these effects is as follows: a 40 pmol/liter increase on serum insulin between entry and the third trimester (see Table 2
) was associated with an increase of 0.36 mU/mg Hb in GPx activity, amounting to 10% of the total increase in GPx (+3.5 mU/mg Hb) between entry and the third trimester. Relationships of a similar magnitude were observed for the total increase in GPx activity with HOMA IR (
10%) and C-Peptide (13%) but with glucose the increase in GPx activity was less 2.7%.
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Table 4 shows that energy-adjusted dietary fat intake was positively correlated with GPx activity during the third trimester (P < 0.01). More interestingly, dietary PUFAs (P < 0.03), n-3 FAs (P < 0.03) and n-6 FAs (P < 0.01) were all significantly correlated with GPx activity during the third trimester, whereas only n-3 FAs was correlated with GPx at entry (P < 0.001). The positive relationship remained when multiple regression models (adjusted for energy, age, pregravid BMI, parity, ethnicity, and cigarette smoking) were performed for the data of the third trimester (ß ± SE were 0.045 ± 0.012, P < 0.001, 0.104 ± 0.04, P < 0.03, 0.820 ± 0.022, P < 0.03, and 0.106 ± 0.05, P < 0.03 for fat, PUFAs, n-3 FAs, and n-6 FAs, respectively).
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There were significant differences in the adjusted means of GPx activity among the three ethnic groups at both entry and during the third trimester (Fig. 1). African-American women had higher levels of GPx activity at entry (25.87 ± 0.58 mU/mg Hb), compared with Caucasians (20.58 ± 1.02 mU/mg Hb, P < 0.0001) and Hispanics (22.28 ± 0.53 mU/mg Hb, P < 0.0001). Similar results were obtained during the third trimester comparing African Americans with Caucasians (28.76 ± 0.59 vs. 24.53 ± 1.03 mU/mg Hb, P < 0.001) and Hispanics (26.21 ± 0.56 mU/mg Hb, P < 0.01). Higher GPx activity for African Americans persisted when each of the four metabolic variables was included in the model. In addition, compared with the others, African-American women had statistically significantly higher intakes of dietary fat (all variables were expressed as percentage of energy intake; P < 0.01), PUFAs (P < 0.03), n-3 FAs (P < 0.03), and n-6 FAs (P < 0.03) (Table 5).
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Discussion |
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Changes in GPx activity and metabolic measures during pregnancy
GPx, a selenium-dependent enzyme, reduces hydrogen peroxide and organic peroxides and leads to the oxidation of glutathione (GSH). Oxidized GSH is reduced by GSH reductase in the presence of nicotinamide adenine dinucleotide phosphate (reduced) and then regenerates GSH. Along with the other antioxidant enzymes, GPx protects cells and tissues from damage caused by reactive oxygen species by helping to maintain balance between prooxidant and antioxidant forces (2, 22).
Normal pregnancy is accompanied by a high metabolic demand and elevated requirements for tissue oxygen, which results in increased oxidative stress and antioxidant defenses (1). We found that GPx activity increased significantly by the third trimester of pregnancy. Prior studies, often involving small numbers of women, found lipid peroxides, such as thiobarbituric acid reactive substances, and various low-molecular-weight antioxidants (such as vitamin E, erythrocyte thiol levels, and iron-binding capacity) increase significantly in the maternal circulation (1, 12, 13, 22, 23). Although increased GPx activity, as a compensatory response to a high lipid peroxide level in pregnancies complicated by hypertensive disorders, has been reported, information on the changes of GPx activity during normal pregnancy has been conflicting (1, 23, 24). Loverro et al. (23) reported that healthy pregnant women (n = 10) had increased thiobarbituric acid reactive substance concentration and the superoxide dismutase activity, but GPx showed no change between the first, second, and third trimesters. In another study, however, GPx activity decreased between the 20th and 30th wk of gestation (13). Possible reasons for failing to observe an increase in GPx activity might be the small sample size, various stages of pregnancy, and/or lack of comparability among multiple samples. The enhanced maternal GPx activity observed in our study population suggests a response of antioxidant defenses to pregnancy-induced increases in oxidative stress.
Our results are consistent with previous findings that showed decreased insulin sensitivity during normal pregnancy (10). During the third trimester, fasting serum insulin concentrations, a hallmark of insulin resistance, along with HOMA IR, an indicator of basal insulin to glucose interaction, were increased, compared with entry data. Fasting plasma C-peptide levels were also markedly higher. C-peptide level has been interpreted as an indicator of insulin secretion because it cosecreted on an equimolar basis with insulin from the ß-cell and is not extracted or metabolized by the liver (25).
Association among GPx activity, insulin resistance, and insulin production
Our data also demonstrated that insulin resistance and insulin production (indicated by plasma C-peptide levels) were positively associated with maternal GPx activity. Information on glucose metabolism and GPx activity in normal pregnant women is limited. To our knowledge, ours is the first to document this relationship during normal pregnancy.
Hyperglycemia is a widely known cause of enhanced plasma free radical concentrations and induces oxidative stress. For example, elevated levels of GPx and SOD and products of lipid peroxidation were found in streptozotocin-induced hyperglycemic rats as well as rat pancreatic islet cells incubated in a high-glucose medium (4, 26, 27). High glucose concentrations were associated with increased activity of GPx and other antioxidants in human endothelial cells as well (3). We observed an increase in GPx activity in normoglycemic pregnant women. This suggests that an elevated glucose level may not be the only factor to induce the oxidative stress.
It has been reported that hyperinsulinemia and insulin resistance without diabetes, e.g. in obesity, during pregnancy and in the presence of excess hormones that act as insulin antagonists are associated with increases in lipid peroxidation products and oxidative stress (6, 7, 28). Patients with pregnancy-induced hypertension, preeclampsia, or polycystic ovary syndrome, all of which are associated with an increase in insulin resistance, have elevated antioxidant enzyme activity and lipid peroxide products (23, 24, 29, 30). Oxidative stress can further impair insulin secretion and action by activating a number of cellular stress-sensitive signaling pathways (6, 7, 28, 31). The participants in the current study had an increase in insulin resistance by the third trimester that was compensated by hyperinsulinemia, thus maintaining normal glucose tolerance. The positive relationship between GPx and the markers of insulin resistance and insulin production suggests that there might be a link between pregnancy-induced oxidative stress, insulin resistance, and ß-cell function.
Relationship between GPx activity and dietary intakes of fat and PUFAs
PUFAs are susceptible to oxidation, and the resulting products may be toxic to the cells (32). A number of studies have been conducted to investigate the effect of lipid on the extent of oxidative damage (7, 8, 9). Paolisso and Giugliano (7) observed in type 2 diabetic patients that increases in plasma nonesterified fatty acid concentrations gave rise to significant increases in plasma malondialdehyde concentrations. One report found that n-3 FAs enhanced the GPx activity in human platelets (8); another showed that human vascular endothelial cells incubated with fish oil enriched with eicosapentaenoic acid and docosahexaenoic acid had increased concentrations of lipid peroxidation products and GPx activity (9). These results suggest that increased GPx activity may be a response to the oxidative stress generated by the relatively high concentrations of PUFAs.
Although data showed in Spanish population (n = 44) that selenium intake from a 72-h recall and a food frequency questionnaire for dietary assessment was correlated with whole-blood GPx activity (r = 0.42 and 0.26 for each) (32), little is known about whether dietary fat intake is associated with GPx activity in human pregnancy. Our data showed that dietary fat and PUFAs including n-3 FAs and n-6 FAs intake were positively correlated with maternal GPx activity, which is consistent with prior in vitro studies (7, 8, 9).
Ethnic differences in GPx activity, dietary fat, and PUFA intake
Studies have revealed significant differences in the prevalence of obesity, diabetes, and metabolic disorders among adults, children, and pregnant women from different ethnic backgrounds (33, 34, 35). Our previous work showed ethnic differences in insulin production and resistance during normal pregnancy (36). There are no prior data on ethnic differences in GPx activity in normal pregnant women. Our research suggests that African Americans have significantly higher GPx activity throughout pregnancy, compared with Hispanics and Caucasians. African Americans also had significantly greater dietary fat and PUFA intakes. What we do not know is whether there are differences in plasma FFA concentrations and lipid peroxides between African Americans and other ethnic groups. Baylin et al. found there was a good correlation between adipose tissue PUFAs and dietary fat intake in healthy nonpregnant men and women (37). Plasma FFA composition is responsive to total dietary fat content (38). Thus, ethnic differences in dietary fat intake, antioxidant status, and glucose metabolism may play a role in developing complications or adverse outcomes during pregnancy.
It is also important to identify the limitations in this study. First, our measure was an antioxidant enzyme and an indirect indicator of oxidative stress. Data have showed that increased GPx activity implicates an enhanced turnover of GSH in response to high levels of lipid peroxide (1, 2). Second, we used an indirect index HOMA IR to estimate insulin resistance instead of euglycemic-hyperinsulinemic clamp because clamp studies are not feasible under conditions of an epidemiologic study. However, data have shown that HOMA IR is strongly correlated with estimates from the hyperinsulinemic clamp; this has been validated in pregnant women (20). Finally, with the measurements of biomarkers, we were able only to determine associations but unable to specify causality among the parameters of interest: insulin resistance, GPx activity, and dietary fat intake during pregnancy. The investigation of the underlying mechanism is important and would be more informative.
In conclusion, we have demonstrated that normal pregnancy is associated with increased GPx activity and insulin resistance. There are ethnic differences in antioxidant response and dietary fat intake. The positive influences of markers of insulin resistance, insulin production, and dietary fat (specifically PUFAs) intake on GPx activity suggest a potential link among diet, insulin resistance, and antioxidant status during pregnancy. These factors may contribute to the increased levels of lipid peroxidation and oxidative stress during pregnancy as well.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMI, Body mass index; FFA, free fatty acid; GPx, glutathione peroxidase; GSH, oxidation of glutathione; Hb, hemoglobin; HOMA IR, homeostasis model of assessment for insulin resistance; n-3 FA, n-3 fatty acid; n-6 FA, n-6 fatty acid; PUFA, polyunsaturated fatty acid.
Received March 28, 2003.
Accepted September 8, 2003.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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in maternal and fetal blood of rats with streptozotocin-induced diabetes: evidence of lipid peroxidation. Am J Obstet Gynecol 183:1035–1040[CrossRef][Medline]
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