Oxidative Stress and Menopausal Status: The Coronary Artery Risk Development in Young Adults Cohort Study

Abstract

Background:

Low endogenous estrogen concentrations after menopause may contribute to higher oxidative stress and greater cardiovascular disease (CVD) risk. However, differences in oxidative stress between similarly aged premenopausal and postmenopausal women are not well-characterized on a population level. We hypothesized that urinary isoprostane concentrations, a standard measure of systemic oxidative stress, are higher in women who have undergone menopause compared to premenopausal women.

Methods and Results:

We examined differences in urinary 8-isoprostane (iPF_2α-III) and 2,3-dinor-8-isoprostane (iPF_2α-III-M) indexed to urinary creatinine between 279 postmenopausal and 196 premenopausal women in the Coronary Artery Risk Development in Young Adults (CARDIA) study, using linear regression with progressive adjustment for sociodemographic factors and traditional CVD risk factors. Unadjusted iPF_2α-III-M concentrations were higher among postmenopausal compared to premenopausal women (Median [25th, 75th percentile]: 1762 [1178, 2974] vs. 1535 [1067, 2462] ng/g creatinine; p = 0.01). Menopause was associated with 25.5% higher iPF_2α-III-M (95% confidence interval [6.5–47.9]) adjusted for age, race, college education, and field center. Further adjustments for tobacco use (21.2% [2.9–42.6]) and then CVD risk factors (18.8% [0.1–39.6]) led to additional partial attenuation. Menopause was associated with higher iPF_2α-III in Black but not White women.

Conclusions:

We conclude that postmenopausal women had higher oxidative stress, which may contribute to greater CVD risk. ClinicalTrials.gov Identifier: NCT00005130.

Introduction

Observational studies demonstrate that premenopausal women have considerably lower risk of cardiovascular disease (CVD) compared to similarly aged men.¹ However, this sex-associated protection diminishes with age, and older women exhibit similar susceptibility to CVD and its risk factors as older men.^2,3 Potential mechanisms for accelerated CVD risk in aging women are incompletely understood but physiologic changes of menopause have been invoked. One pathway which may link menopause to cardiovascular health is loss of sex hormone modulation of oxidative stress.⁴

Oxidative stress, perturbation of the balance between oxidative molecules such as reactive oxygen species (ROS) and the body's antioxidant defense system, has been implicated in the pathophysiology of many diseases.⁵ Isoprostanes are prostaglandin-like compounds, which can be produced nonenzymatically when ROS react with arachidonic acid, and are commonly used to assess oxidative stress of the internal milieu. Few studies have investigated associations between menopausal status and isoprostanes and the findings to date have been inconsistent.^6,7

Furthermore, these studies are typically conducted among cohorts of women with a wide age range, making it difficult to disentangle correlates of menopause from effects of aging itself and secular trends of potential confounders.^7,8 Finally, while 8-isoprostane (iPF_2α-III) has been the most commonly assayed isoprostane in the past, several metabolites of iPF_2α-III, including 2,3-dinor-8-isoprostane (iPF_2α-III-M), have higher concentrations in vivo and could be more sensitive markers for systemic oxidative stress.^7,9

We measured urinary concentrations of iPF_2α-III and iPF_2α-III-M in a cohort of premenopausal and postmenopausal women with a narrow age range. We hypothesized that postmenopausal status is associated with higher oxidative stress, implying a possible mechanism which might contribute to greater CVD risk.

Methods

Study population

The Coronary Artery Risk Development in Young Adults (CARDIA) study is a community-based, multicenter, observational, longitudinal cohort study sponsored by the National Heart, Lung, and Blood Institute. CARDIA enrolled 5115 women and men between 18 and 30 years of age who were free from CVD from four centers (Birmingham, AL; Oakland, CA; Chicago, IL; and Minneapolis, MN) at baseline in 1985–86.¹⁰ The institutional review boards of all participating study sites approved the study, and all participants provided written informed consent.

This cross-sectional analysis was conducted among a subset of female participants included in an ancillary study to CARDIA through which urinary isoprostanes at the year 25 follow-up examination (2010–2011) were measured. This ancillary study was performed as part of/and funded by the “Go Red for Women” Strategically Focused Research Network grant awarded by the American Heart Association. Full description of the inclusion and exclusion criteria for the ancillary study is listed in the Supplementary Data S1. In short, our participant group comprised all CARDIA women between 48 and 55 years old at the year 25 visit who had urinary isoprostanes available. This CARDIA ancillary study excludes women with (1) undetermined menopausal status based on questionnaire data, (2) use of hormone replacement therapy or oral contraceptives, (3) history of hysterectomy, and (4) pregnancy or breastfeeding.

For this analysis, we also excluded women with estimated glomerular filtration rate <60 mL/(min × 1.73 m²) calculated using the serum creatinine-based modification of diet in renal disease study group equation¹¹ (10/485 women), since urine-based assays may not be interpretable in persons with impaired renal function.

Women on hormone therapy who otherwise met the criteria were not included as they are physiologically distinct from the included groups and were not present in sufficient number to form a separate analysis group. We did not exclude the small minority of our population (23 women) who reported history of gynecologic surgery other than hysterectomy (which technically may include bilateral oophorectomy with uterine preservation). Our definition of menopause is robust against misclassifications related to surgically-induced menopause as (1) it correctly encompasses women in long-standing (>12 months) surgical menopause as postmenopausal, and (2) since no participants reported cessation of menses following surgical intervention in the 12 months preceding their examination date, no woman undergoing acute changes of surgical menopause was misclassified as premenopausal because of the 12-month rule. Based on the above criteria, 196 premenopausal and 279 postmenopausal women were included (Supplementary Fig. S1).

Measurement of exposure variables

Standardized questionnaires, physical examination, and laboratory measures were used to assess exposure variables.¹⁰ Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. All participants were asked to fast for 12 hours before each clinic visit. Blood pressure was measured using the Omron device (Omron Healthcare, Inc., Lake Forest, IL) and calibrated to the random-zero measures. Plasma total cholesterol, high-density lipoprotein cholesterol (HDL-c), and triglyceride levels were measured using enzymatic methods. CARDIA physical activity questionnaire was used to measure the frequency of participation in 13 different categories of recreational sports and activities in the past 12 months. Physical activity scores accounting for frequency of participation and intensity of activity were calculated (metabolic equivalent of task multiplied by the sum of months of infrequent plus 3 × months of frequent participation in the prior year for each type of activity) and reported as “exercise units” (EU).¹²

Diabetes was defined as fasting glucose ≥126 mg/dL or treatment with hypoglycemic medications. Menopause was defined as >12 months since last menstruation. Perimenopause (for sensitivity analysis) was defined as cessation of menstruation for ≤12 months or subjective change in the interval between periods in the past year (96 premenopausal women met these criteria).

Urinary isoprostane measurements

Urinary concentration of iPF_2α-III and iPF_2α-III-M was measured at the Atherosclerosis Clinical Research Laboratory at Baylor College of Medicine (Houston, TX). Samples underwent sequential washing with solvent mixtures on mixed anion solid phase exchange columns for subsequent measurement of isoprostanes by gas chromatography–mass spectrometry.¹³ Assay variance was assessed using two pools of quality control samples for each isoprostane entity and yielded intra-assay coefficient of variation (CV) of 5%–7% and interassay CV of 21%–24% (Supplementary Table S1). To account for variations in urine concentration, each isoprostane measurement was indexed to urinary creatinine (measured via Jaffe rate method).¹⁴

Statistical analysis

Population characteristics were stratified by menopausal status. Quantitative measurements with normal distribution were reported as mean (standard deviation), those with non-normal distribution were reported as median [quartile 1, quartile 3], and categorical characteristics were reported as number (percentage). Unadjusted differences between these groups were tested using Student's t, rank sum, or chi square tests, respectively. Fasting serum triglycerides and creatinine indexed iPF_2α-III and iPF_2α-III-M values were log-transformed due to right skewed distribution. Cross-sectional associations between urinary isoprostanes (as dependent variables in separate models) and menopausal status (independent variable) were explored using multivariable linear regression.

To differentiate associations of menopause from effects of behavioral factors (such as smoking), or biological factors potentially affected by menopause (such as lipids), or other socioeconomic, demographic, and cardiovascular health confounders, models were progressively adjusted: Model 1 adjusted for age, race, college attainment, and study center; model 2 included covariates from model 1 plus smoking status (current, former, or never) and cumulative pack-years; and model 3 additionally adjusted for BMI, diabetes, systolic blood pressure, use of medication for hypertension, total cholesterol, HDL-c, fasting triglyceride (log transformed), use of lipid-lowering medication, serum creatinine, and physical activity. Current smoking status and cumulative pack-years were included simultaneously for more precise and quantitative adjustment for tobacco use. Interaction by race was assessed for each model, and analysis stratified by race was performed when statistically significant interaction between an isoprostane entity and race was found.

As secondary analyses, we performed stepwise regression with backward selection (p < 0.10 threshold) to get a more parsimonious set of covariates, and also constructed a new set of models in which menopausal status was divided into three categories (premenopausal/perimenopausal/postmenopausal).

Since log-transformed isoprostane concentrations were used as dependent variables, model results are presented as exponentiated β-coefficients and reflect geometric ratios (ratio of isoprostanes in one group compared to the other). Ratios >1 indicate a positive relationship, and ratios <1 indicate an inverse relationship. Two-sided p-values <0.05 were considered statistically significant. Analyses were performed on Stata v15.1 (StataCorp LP, College Station, TX). Author D.V. had full access study data and takes responsibility for its integrity and analysis.

Results

Population characteristics

Characteristics of the sample are shown in Table 1. Postmenopausal women were slightly older (difference in mean age <2 years), more likely to use cholesterol lowering medications (16.3% vs. 8.7%; p = 0.02), and had higher total cholesterol concentration (200.9 mg/dL vs. 193.6 mg/dL; p = 0.030) compared to premenopausal women. There were trends toward greater use of blood pressure medications (p = 0.08) and active tobacco use (p = 0.09, current vs. never/former smokers) among postmenopausal compared to premenopausal women.

Table 1.

Participant Characteristics at Coronary Artery Risk Development in Young Adults Year 25 Examination by Menopausal Status

	Premenopausal (n = 196)	Postmenopausal (n = 279)	p
Age, years	51.2 (2.2)	53.0 (1.9)	<0.001
Age at menopause, years		48.9 (4.0)
Black race	40.3%	37.3%	0.50
Body mass index, kg/m2	30.3 (8.5)	29.7 (7.3)	0.41
≥College degree (%)	61.0%	57.7%	0.47
Smoking status (%)			0.17
Never	65.1%	58.8%
Former	24.6%	25.6%
Current	10.3%	15.7%
Cumulative smoking, pack-years	0.7 (2.7)	1.2 (3.8)	0.10
Nonzero cumulative smoking, pack-years (never smokers excluded)	5.2 [3.8, 8.6]	6.3 [3.6, 12.6]	0.72a
Moderate/vigorous physical activity, EUb	313 (230)	298 (231)	0.49
Systolic blood pressure, mmHg	116.1 (15.8)	116.7 (16.4)	0.69
Total cholesterol, mg/dL	193.6 (32.3)	200.9 (38.2)	0.030
HDL cholesterol, mg/dL	64.2 (17.0)	65.8 (17.0)	0.31
Triglycerides, mg/dL	94.8 (66.6)	100.1 (52.2)	0.33
Serum creatinine, mg/dL	0.76 (0.11)	0.74 (0.12)	0.10
Diabetes (%)	6.6%	7.5%	0.71
Antihypertensive medication use (%)	17.3%	24.0%	0.080
Cholesterol lowering medication use (%)	8.7%	16.3%	0.016
Urinary creatinine, mg/dL	104.8 (76.3)	104.6 (68.9)	0.97
Urinary iPF2α-III, ng/g creatinine	873 [529, 1318]	892 [593, 1455]	0.38
Urinary iPF2α-III-M, ng/g creatinine	1535 [1067, 2462]	1762 [1178, 2974]	0.010

Data presented as mean (SD), median [Q1, Q3], or percentage of participants.

^a This test should be interpreted in the context of the applied conditional requirement (nonzero pack-years only).

^b EU: Exercise units as described previously.¹²

HDL, high-density lipoprotein.

Urinary isoprostanes and menopausal status

iPF_2α-III-M concentrations were higher in postmenopausal compared to premenopausal women (1762 [1178, 2974] vs.1535 [1067, 2462] ng/g creatinine, respectively; p = 0.010). In contrast, there was no difference in urinary iPF_2α-III by menopausal status (892 [593, 1455] vs. 872 [529, 1318] ng/g creatinine, respectively; p = 0.38) (distributions in Fig. 1).

FIG. 1.

Distribution of urinary isoprostanes by menopausal status. Both iPF_2α-III and iPF_2α-III-M showed right skewness and thus have been plotted on the logarithmic scale. iPF2α-III, 8-isoprostane; iPF_2α-III-M, 2,3-dinor-8-isoprostane.

Linear regression models were used to study associations between isoprostanes and menopause with progressive adjustments. iPF_2α-III was not associated with menopause among the entire cohort (Fig. 2a). However, there was a significant interaction between iPF_2α-III and race (unadjusted p = 0.04) so analysis for this variable was repeated after stratification by race. When stratified by race, menopausal status was associated with higher iPF_2α-III in Black women but not in White women (Fig. 2b). This association was robust against adjustments, and postmenopausal Black women had 33% higher iPF_2α-III compared to premenopausal Black women (confidence interval [95% CI] 2.3–71.7).

FIG. 2.

(a) Adjusted associations between menopause and iPF_2α-III. Exponentiated β-coefficients are equivalent to the ratio of analyte of interest in postmenopausal to premenopausal women after adjustment. Ratios >1 represent positive correlation, and ratios <1 represent inverse correlation. Models were progressively adjusted as follows: Model 1—age, race, college attainment, field center; Model 2—Model 1 + smoking status (current, former, or never), cumulative pack-years; Model 3—Model 2 + BMI, diabetes (fasting glucose ≥126 mg/dL or use of diabetes medication), systolic blood pressure, use of medication for hypertension, total cholesterol, HDL cholesterol, fasting triglycerides (log transformed), use of lipid lowering medication, serum creatinine, and physical activity. (b) Adjusted associations between menopause and iPF_2α-III stratified by race. Exponentiated β-coefficients are equivalent to the ratio of analyte of interest in postmenopausal to premenopausal women after adjustment. Ratios ≥1 represent positive correlation and ratios ≤1 represent inverse correlation. Models were progressively adjusted as follows: Model 1—age, college attainment, field center; Model 2—Model 1 + smoking status (current, former, or never), cumulative pack-years; Model 3—Model 2 + BMI, diabetes (fasting glucose ≥126 mg/dL or use of diabetes medication), systolic blood pressure, use of medication for hypertension, total cholesterol, HDL cholesterol, fasting triglycerides (log transformed), use of lipid lowering medication, serum creatinine, and physical activity. p-Values for interaction between race and menopause were 0.04 (unadjusted), 0.03 (model 1), 0.03 (model 2), and 0.01 (model 3). BMI, body mass index; CI, confidence interval; HDL, high-density lipoprotein.

In a univariate model, iPF_2α-III-M was 28.9% higher among postmenopausal compared to premenopausal women (Fig. 3). In the minimally adjusted model (age, race, college attainment, and field center; N = 474) iPF_2α-III-M remained 25.5% higher in the postmenopausal group. After additional adjustments for tobacco use (N = 468) the association between menopause and iPF_2α-III-M was attenuated to 21.2% but remained statistically significant, and similarly, further adjustment for traditional CVD risk factors (N = 464) resulted in additional partial attenuation to 18.8% (95% CI after all adjustments: 0.1–39.6). Interactions between iPF_2α-III-M and race were not statistically significant (unadjusted p = 0.10) so race-stratified analysis was not performed.

FIG. 3.

Adjusted associations between menopause and iPF_2α-III-M. Exponentiated β-coefficients are equivalent to the ratio of analyte of interest in postmenopausal to premenopausal women after adjustment. Ratios >1 represent positive correlation, and ratios <1 represent inverse correlation. Models were progressively adjusted as follows: Model 1—age, race, college attainment, field center; Model 2—Model 1 + smoking status (current, former, or never), cumulative pack-years; Model 3—Model 2 + BMI, diabetes (fasting glucose ≥126 mg/dL or use of diabetes medication), systolic blood pressure, use of medication for hypertension, total cholesterol, HDL cholesterol, fasting triglycerides (log transformed), use of lipid lowering medication, serum creatinine, and physical activity.

In our secondary analysis with backwards selection, iPF_2α-III-M maintained its statistically significant association with menopausal status and was retained in the final trimmed model (Supplementary Table S2; exponentiated β [95% CI] 1.205 [1.037–1.399]) alongside BMI, smoking status, diabetes, serum creatinine, plasma HDL-c, and triglycerides.

Sensitivity analysis

In a sensitivity analysis, indexing by urinary creatinine was substituted with inclusion of urinary creatinine as an independent variable in the regression models¹⁵; however, the results were essentially unchanged (data not shown). Separation of perimenopausal women into their own category resulted in reduced power particularly in our extensively adjusted models (as a result of smaller sample size in the groups), but the results remained consistent with our findings in the entire cohort and showed that oxidative stress in the perimenopausal group was more similar to the premenopausal group and lower than the postmenopausal group (Supplementary Table S3).

Discussion

We measured in vivo oxidative stress with urinary isoprostanes among premenopausal and postmenopausal women from a well-characterized and relatively healthy community-based cohort. We found higher oxidative stress among postmenopausal compared to the premenopausal women as measured by iPF_2α-III-M. Association between iPF_2α-III and menopausal status varied by race, such that in Black women, menopausal status was associated with significantly higher oxidative stress, but no association was present in White women. To our knowledge this is the first study analyzing both iPF_2α-III and its 2,3-dinor metabolite in menopause. Some metabolites of iPF_2α-III have higher urinary concentration than iPF_2α-III itself, and may be more sensitive markers of oxidative stress.⁷ As iPF_2α-III-M is the product of β-oxidation of iPF_2α-III, it is speculated to be more representative of systemic oxidative stress and less prone to local renal production^9,16; however, differences in metabolism of isoprostanes cannot be ruled out as a potential source of variability in associations.¹⁶

Inclusion of both iPF_2α-III and its metabolite in our study illustrates differences between the two oxidative stress markers for the first time in menopause. In addition, our study is strengthened by a narrow age range in our study population and relatively small (1.8 years) difference in the mean age of our groups of interest, which limits confounding by age and secular trends.

We report higher urinary iPF_2α-III-M irrespective of race and higher iPF_2α-III only in Black women, consistent with higher oxidative stress associated with menopausal status. This association persisted after adjustment for sociodemographic factors, smoking, and traditional CVD risk factors although inclusion of these factors did result in partial attenuation. Higher oxidative stress could contribute to increased susceptibility to future CVD events and mortality in these postmenopausal women,^17,18 and isoprostanes themselves have been reported to have detrimental cardiovascular effects as well.¹⁹

Our results add to a sparse and inconsistent body of literature. Previous studies have reported higher concentration of some markers of oxidative stress such as lipoperoxides, malonaldehyde, and 4-hydroxynonenal in postmenopausal women. In addition, low density lipoprotein particles in postmenopausal women have been found to be more susceptible to oxidation compared to premenopausal women^20–22; however, the contrary has also been reported.²³ Moreover, those markers are considered inferior to isoprostanes in assessment of in vivo oxidative stress.²⁴ More recently, a study found low serum free thiols (which are consumed by ROS and therefore reflective of oxidative stress) to be associated with menopause, as well as increased incidence of adverse cardiac events, although this was no longer significant when corrected for age.²⁵

Race differences in oxidative stress markers have been reported previously. Higher BMI has been demonstrated to be associated with higher isoprostanes in White but not Black adults.²⁶ In contrast, oxidative stress in Black women has been found to be associated with insulin sensitivity but not in their White counterparts.²⁷ In a study of blood pressure and markers of oxidative stress, isoprostanes were associated with higher blood pressure in Black participants only, whereas similar associations were reported for 8-hydroxydeoxyguanosine in White participants.²⁸ Our results add to this active area of research about racial differences in oxidative stress mechanisms of CVD.

Only a handful of studies have measured isoprostanes in the context of menopause. In 2002, a small Swedish study failed to show any association between urinary isoprostanes and migraine headaches (its primary goal) but reported significantly higher concentration of iPF_2α-III in postmenopausal compared to premenopausal participants. Similar findings were reported in a study of breast cancer.²⁹ Later, the Shanghai Women's Health Study did not show a difference in iPF_2α-III by menopausal status but found one of its metabolites (which is downstream of iPF_2α-III-M) to be elevated in the postmenopausal subgroup.^7,30 This study included a large sample size, but did not distinguish menopause-specific changes in isoprostanes from age-related trends which were also significant. Another notable study conducted by Sowers et al. found no associations between menopausal status and iPF_2α-III among women 47 to 57 years old but did not test any of iPF_2α-III metabolites.⁶ Due to the limitations of these studies, whether postmenopausal women have higher oxidative stress independent of aging and other confounders has remained ambiguous.

Potential mechanisms that could underlie higher oxidative stress after menopause are also debated, with a commonly cited hypothesis that estrogen may reduce oxidative stress.³¹ However, the Sowers study reported a positive association between estrogens and iPF_2α-III, and a study in another cohort of premenopausal women had similar findings.^6,32 These studies could suggest that increased oxidative stress after menopause may be caused by factors other than low estrogen; however, more population-level research of oxidative stress and sex hormone concentrations—particularly in perimenopausal and postmenopausal women—is required, as effect modification by age has been suggested to result in a pseudo-paradoxical relationship between sex hormones and oxidative stress.³¹ Activated estrogen receptors in the cardiovascular system localize to the cell nucleus and promote the transcription of nitric oxide synthase (NOS). In young and healthy adults this enzyme produces nitric oxide (NO) and contributes to cardioprotective vasodilation.

However, with aging, other cofactors necessary for NOS function are depleted, and the enzyme is suggested to become “uncoupled” from the NO pathway to produce superoxides and other deleterious ROS.³¹ Furthermore, it is important to note that aging could alter the expression of estrogen receptors in the cardiovascular system, and thus, differences in estrogen concentration may not translate to differences in estrogen activity across the age spectrum.³³

We also found borderline significant higher prevalence of current versus former/never smokers in our postmenopausal group, which led to partial attenuation of the association between menopause and oxidative stress. Even though menopause is associated with changes in mood and behavior, it is unlikely that menopausal transition would lead to new tobacco use. In contrast, it is well-documented that smoking is linked to earlier menopause.³⁴ As smoking is also strongly linked to higher oxidative stress, it would be feasible to consider oxidative stress as a potential mediator linking smoking to early menopause. Moreover, a recent study from the same CARDIA cohort found higher plasma isoprostane concentrations to be associated with lower anti-Mullerian hormone levels (suggestive of lower ovarian reserve) particularly in younger women.³⁵

Whether increased oxidative stress is caused by or results from ovarian dysfunction remains unknown; however, the role of oxidative stress in CVD pathophysiology is well-established and previous studies have shown early menopause to be predictive of poor cardiovascular outcomes itself,³⁶ suggesting that oxidative stress could play an important role in cardiovascular health of women, regardless of the directionality of its association with menopause.

We found existing CVD risk factors to be important in explaining some of the differences between the premenopausal and postmenopausal group. For some CVD risk factors such as plasma lipids (noted to be different among our participant groups), studies show a menopause-related acceleration in the rate of change of plasma concentrations not explained by chronological aging.³⁷ Another important consideration is whether menopause could be associated with qualitative changes in CVD risk factors. A recent study suggests that higher HDL-c could be protective in premenopausal women, but deleterious in postmenopausal women.³⁸ This is particularly interesting as HDL particles are thought to be “scavengers” of oxidized lipids and the primary carriers of isoprostanes in the blood.³⁹ Clearly, various CVD risk factors could be associated with oxidative stress and menopause differently, creating a complex and interlinked system which requires further investigation itself.

Our study's strengths include its relatively large, well-characterized sample population and narrow range of age of the participants allowing focus on the perimenopausal time frame. Furthermore, we measured urinary isoprostanes by gas chromatography–mass spectroscopy, which is more accurate than enzyme immunoassays,⁴⁰ and for the first time included iPF_2α-III-M in a study of menopause. Use of urinary measures of isoprostanes rules out in vitro oxidization after sample collection which is crucial when using precollected samples.

Our study also had limitations. Considering the observational and cross-sectional nature of our study, we cannot distinguish which of oxidative stress or menopause precedes the other. Relatively high interassay CV may have contributed to lower power to detect associations for iPF_2α-III. We also did not perform sex-hormone assays and could not determine if the differences in oxidative stress were associated with differences in estrogen concentration. Reproductive criteria (e.g., number of pregnancies), as well as other potential variables of interest such as supplemental antioxidant use, were not explored in this analysis, as the CVD focus of this project required prioritization of cardiovascular factors to avoid overfitting.

We did not differentiate natural menopause from surgical menopause resulting from bilateral oophorectomy with uterine preservation. However, only a small minority of women reported any ovarian surgery without hysterectomy in our group, and therefore, separate analyses for this group were not feasible. Moreover, none of the participants reported cessation of menses after ovarian surgery in the 12 months leading to the examination date; hence, misclassification as a result of recent surgical menopause was also not an issue in our study. Self-reported menopausal status may lead to misclassification; however, internal validation assessments in CARDIA have found this error to be minute. Some studies have suggested that isoprostane concentrations could differ throughout the menstrual cycle, which we did not account for. However, we believe that imposing limitations on phases of the cycle would have lowered the generalizability of our results.

Conclusion

In summary, postmenopausal women had evidence of higher oxidative stress than premenopausal women independent of smoking and other CVD risk factors. This difference was reflected in higher creatinine-indexed concentration of iPF_2α-III-M in postmenopausal women compared to premenopausal women of similar age before and after adjustments for the confounding factors listed above. In contrast, iPF_2α-III did not differ based on menopause status in the overall cohort, which could be related to lower sensitivity of this marker or differences in isoprostane metabolism. However, menopause was associated with higher iPF_2α-III in Black but not White women. Increased oxidative stress could contribute to susceptibility to development of CVD in postmenopausal women.

Disclaimer

The views expressed in this article are those of the authors and do not necessarily represent the views of the National Heart, Lung, and Blood Institute; the National Institutes of Health; or the U.S. Department of Health and Human Services.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was funded by the American Heart Association Go Red for Women Strategically Focused Research Network grant 16SFRN27870000. A.S.H. was awarded funding provided, in part, by Nancy Grasmick, EdD, the Division of Cardiology, Department of Medicine and the Office of Medical Student Affairs of Johns Hopkins School of Medicine to conduct 1 year of dedicated research as a medical student. E.D.M. and D.Z. are additionally funded by the Blumenthal Scholars Award in Preventive Cardiology at Johns Hopkins University. The Coronary Artery Risk Development in Young Adults Study (CARDIA) is supported by contracts HHSN268201800003I, HHSN268201800004I, HHSN268201800005I, HHSN268201800006I, and HHSN268201800007I from the National Heart, Lung, and Blood Institute (NHLBI). Funding sources were not involved in study design, analysis, interpretation of data, or the writing of the report.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Figure S1