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Published in final edited form as: Life Sci. 2020 Oct 15;264:118590. doi: 10.1016/j.lfs.2020.118590

Changes in erythrocyte membrane epoxyeicosatrienoic, dihydroxyeicosatrienoic, and hydroxyeicosatetraenoic acids during pregnancy

Selina T Somani 1, Maxwell Zeigler 2, Emily E Fay 3, Maggie Leahy 1, Bethanee Bermudez 1, Rheem A Totah 2, Mary F Hebert 1,3
PMCID: PMC7755749  NIHMSID: NIHMS1639598  PMID: 33069736

Abstract

Aims

Pregnancy is associated with numerous changes in physiological and metabolic processes to ensure successful progression to full term. One such change is the alteration of arachidonic acid (AA) metabolism and formation of eicosanoids. This study explores the changes in AA metabolites formed through the cytochrome P450 mediated pathway to epoxyeicosatrienoic (EET), dihydroxyeicosatrienoic (DHET), and hydroxyeicosatetraenoic (HETE) acids which have been implicated in blood pressure regulation and inflammatory responses that are important for a healthy pregnancy.

Main methods

The study determines circulating levels of EETs, DHETs and HETEs extracted from erythrocyte membranes and measured by mass spectroscopy during the progression of a normal pregnancy. Blood samples, from 25 women, were collected at three time points including 25-28 weeks gestation, 28-32 weeks gestation, and the non-pregnant control at 3-4 months postpartum.

Key Findings

Results demonstrate that healthy pregnancy is associated with significant increases in 8,9-DHET, 11,12-DHET and 14,15-DHET and a decrease in trans 8,9-EET during 28-32 weeks gestation compared to 3-4 months postpartum. These differences are likely due to several mechanisms including an increase in soluble epoxide hydrolase activity, a decrease in glutathione conjugation, and altered cytochrome P450 enzyme expression and/or activity that occurs during pregnancy.

Significance

Metabolism of AA through the cytochrome P450 pathway generates physiologically important eicosanoids that could play an important role in the progression of a healthy pregnancy. Establishing the changes that occur during normal pregnancy may, in the future, help in early detection of pregnancy complications including pre-eclampsia.

Keywords: Pregnancy, arachidonic acid, epoxyeicosatrienoic acid, dihydroxyeicosatrienoic acid, hydroxyeicosatetraenoic acid, soluble epoxide hydrolase

Graphical Abstract

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1. Introduction

During pregnancy, the fetus is tolerated within the mother’s uterus while simultaneously, the body can defend against pathogens through upregulation of neutrophils, macrophages, and C-reactive protein [1,2]. In many women, the severity of autoimmune diseases is decreased during pregnancy [2]. At the same time, pregnant women are at an increased risk of developing complications from some infections including herpes simplex virus (HSV), malaria, and influenza [3].

Polyunsaturated fatty acids, like AA and their metabolites, are major mediators in vascular tone and immunomodulatory processes [4]. The AA cascade has three major branches, including the cyclooxygenase, thromboxane, and cytochrome 450 (CYP) pathways. The cyclooxygenase and thromboxane pathway’s effects on pregnancy progression and termination have been extensively studied [5,6]. In contrast, the effect of the cytochrome P450 (CYP) pathway on the progression of pregnancy is under studied, although there are reports that metabolism is potentially altered by pregnancy [7]. Previous work suggests that 5-,15- and 20-HETE (hydroxyeicosatetraenoic); 5,6-, 8,9- and 11,12-DHET (dihydroxyeicosatrienoic); as well as 5,6-, 11,12- and 14,15-EET (epoxyeicosatrienoic) are increased during pregnancy, but these studies are limited by design issues [8-10]. Interestingly, the number of changes observed and the magnitude of effects on the AA cascade are greater in pathogenic conditions during pregnancy such as preeclampsia [8,11]. Determining the changes in both normal and oxidized fatty acids during pregnancy may aid in better understanding the immunomodulation that occurs during pregnancy. Importantly, it will establish the norms for later evaluating alterations that occur in important pathologic conditions like preeclampsia. The purpose of this study is to characterize the effects of normal pregnancy progression on erythrocyte membrane EETs, DHETs, and HETEs. We hypothesize that altered enzyme activity during normal pregnancy will alter the concentrations of EETs, DHETs and HETEs.

2. Materials and Methods

Optima grade solvents including methanol, ethyl acetate, acetonitrile (ACN), water, chloroform, 2-propanol, acetic acid, triphenylphosphine (TPP), NaCl, HCl, KOH, bicinchoninic acid (BCA) assay, and 10x concentrated phosphate buffered saline (PBS) were purchased from Fisher Scientific (Waltham, MA, USA) and used without further purification. Butylated hydroxytoluene (BHT) was purchased from MP Bio (Santa Ana, CA, USA). The 1-Hydroxybenzotriazole (HOBT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The (±) 5,6-dihydroxy-8Z,11Z,14Z-eicosatrienoic acid, (±) 8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid, (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid, (±) 14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid, (±) 14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic-d11 acid (±) 5,6-epoxy-8Z,11Z,14Z-eicosatrienoic acid, (±) 5,6-epoxy-8Z,11Z,14Z-eicosatrienoic-d11 acid, (±) 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic acid, (±) 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic-d11 acid, (±) 11,12-epoxy-5Z,8Z,14Z-eicosatrienoic acid, (±) 14,15-epoxy-5Z,8Z,11Z-eicosatrienoic acid, (±) 14,15-epoxy-5Z,8Z,11Z-eicosatrienoic-d11 acid, (±) 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid, (±) 8-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid, (±) 9-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid, (±) 11-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid, (±) 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-d8 acid, (±) 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid, 19S-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, and 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The 1-(4-Aminomethyl) phenyl) pyridine-1-ium chloride (AMPP) was purchased from Oxchem Corporation (Wood Dale, IL, USA). The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from TCI (Tokyo, Japan). Ultra-high purity nitrogen gas (5.0 grade) was purchased from Praxair (Danbury, CT, USA). The 96-well polypropylene cap mats as well as the 350 μL and 2 mL polypropylene 96-well plates were purchased from NUNC (Rochester, NY, USA). The 96-well 0.45 μm hydrophilic polytetrafluoroethylene (PTFE) filters were purchased from EMD Millipore (Darmstadt, Germany). Plate dryer, positive pressure manifold, and 96-well Evolute Express ABN solid phase extraction (SPE) 30 mg plate were purchased from Biotage (Uppsala, Sweden).

2.1. Study Design

EETs, DHETs, and HETEs from the CYP mediated pathways of AA were extracted from red blood cell (RBC) membranes and measured in 25 pregnant women at 25 to 28 weeks gestation, 28 to 32 weeks gestation, and 3-4 months postpartum. For each subject, there was 3-4 weeks between the 2 pregnancy study days. All 25 subjects completed the 3 study days and each subject served as their own control.

2.2. Subject Selection Criteria

This study included 25 pregnant women, 27 to 41 years of age with singleton pregnancies. Participants were excluded for fever, cough, known kidney or liver disease, diabetes, body mass index (BMI) > 30 kg/m2, and psychiatric illnesses requiring medication. The study protocol was approved by the University of Washington Institutional Review Board and conducted in accordance with their guidelines.

2.3. Sample Collection and Processing

Blood samples were collected in foil wrapped lavender top tubes (containing EDTA as an anticoagulant) one hour following breakfast. Each subject was allowed to choose their own breakfast. Samples were placed on wet ice prior to processing. The samples were then centrifuged within 10 minutes and processed in a laboratory with ultraviolet (UV) light filters covering the lights. The RBC membranes were washed twice with PBS and then stored at −80° C for up to 19 months until analysis. EET, DHET and HETE acid samples have been shown to be stable for up to 10 years when stored at −80° C when the samples have not been previously thawed [12]. Serum was assayed for serum creatinine concentration. Urine was collected over 4 hours and stored at 4° C until sample collection was completed, then analyzed for urine creatinine concentration.

2.4. Eicosanoid Extraction Protocol

Eicosanoids were extracted from RBC membranes using an adapted method previously published for human plasma [12]. Eicosanoid extraction was carried out utilizing a 2 mL capacity 96-well plate to increase throughput with pooled RBC samples placed randomly on the plate. The extraction was also carried out in low light and samples were stored on ice whenever possible to minimize autoxidation. Total lipids were extracted using a modified Bligh and Dyer protocol [13]. Briefly, RBC samples were diluted 1:3 in a 0.9% NaCl solution and 20 μL of this solution was added to each well of a 2 mL 96-well plate containing 10 μL of 25 ng/mL internal standard mix, 2 μL of 50 mM BHT/5 mM TPP antioxidant mix, 170 μL of 0.9% NaCl, 250 μL of chloroform, and 500 μL of methanol. The plate’s head space was flooded with nitrogen gas, covered with a cap mat, and vortexed for 2 minutes. Chloroform (250 μL) and 250 μL of 0.9% NaCl with 0.1% acetic acid were then added and the plate was again flooded with nitrogen gas, covered with a cap mat, and vortexed. The samples were then centrifuged for 10 minutes at 4,500 × g and the chloroform layer was collected and evaporated to dryness under nitrogen gas.

Fatty acids were freed from phospholipids by saponification. Two-hundred microliters of 2-propanol and 200 μL of 1.0 M KOH were added to each sample. The head space was capped with nitrogen gas, a cap mat was added, then the samples were briefly vortexed and incubated in a water bath at 37° C for 45 minutes. The samples were placed on ice where 200 μL of 1.0 M HCl and 400 μL of PBS were added.

After saponification, the fatty acids were purified by solid phase extraction. An SPE cartridge was conditioned with 1 mL ethyl acetate, 1 mL methanol, and 1 mL PBS. The samples were then loaded onto the SPE cartridge and then washed with 3 mL of a solution of 95% water and 5% ACN. The cartridge was then dried under nitrogen gas. After drying, the fatty acids were eluted into a 96-well plate with 500 μL of methanol followed by 1 mL ethyl acetate and the eluent dried under nitrogen gas

The fatty acids were derivatized by subsequently adding 30 μL of ACN, 10 μL of 640 mM EDC, 20 μL of 5 mM HOBT, and then 20 μL of AMPP. The 96-well plate had its head space filled with nitrogen gas and capped with a cap mat and the derivatization reaction proceeded for 30 minutes in a 60° C water bath. The samples were then cooled at 4° C before centrifugal filtration using a 0.45 μm hydrophilic PTFE filter and analyzed by LC-MS/MS as described previously [12]. The protein content of the RBCs was determined by BCA assay [14]. Each subject’s samples were run on a single 96-well plate to minimize variability. Quality check sample from the same pool of RBC were included on each plate to assess variability [12]. Inter-plate variabilities were 1.8% for 8,9 DHET, 12.0% for 11,12 DHET, 6.4% for 14,15 DHET and 27.3% for 8,9-trans-EET.

2.5. Data Analysis

Reported fatty acid content are unit-less peak area ratios for each fatty acid divided by the corresponding internal standard. Those peak area ratios were then normalized to total RBC membrane protein.

Creatinine clearance was estimated by CrCl = (Uv•UCr)/(SCr•time), where Uv is urine volume, UCr is urine creatinine, and SCr is serum creatinine.

Paired, two-sided, Students T-test was used to compare the normalized EET, DHET, and HETE fatty acid content as well as creatinine clearance between gestational weeks 25-28 and 28-32, gestational weeks 25-28 and postpartum, and gestational weeks 28-32 and postpartum. P values < 0.05 were considered statistically significant. Adjustments for multiple comparisons was not conducted.

3. Results

Twenty-five subjects (18 Caucasian, 3 Asian, 1 Black, 1 Hawaiian and 2 Caucasian/Asian) participated in this study and completed all 3 study days. Mean (± SD) subject’s age, height and weights on all 3 study days are reported in Table 1. Body weights averages were significantly higher during pregnancy (25-28 weeks gestation 68.2 ± 9.3 kg p=0.008 and 28-32 weeks gestation 70.1 ± 9.5 kg p=0.00003) than postpartum (65.4 ± 8.6 kg). Creatinine clearance was significantly higher during 25-28 weeks gestation (209 ± 52 mL/min, p=4E-09) and 28-32 weeks gestation (217 ± 49 mL/min, p=2E-11) compared to postpartum (136 ± 29 mL/min).

Table 1.

Subject Demographics

Weight
(kg)		 Age
(years)		 Height
(cm)
25-28 weeks Gestation 28-32 weeks Gestation Postpartum
68.2 ± 9.3** 70.1 ± 9.5** 65.4 ± 8.6 32.6 ± 3.7 165.7 ± 6.5
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Results reported as mean ± SD.

**

p < 0.01 as compared to postpartum.

Normalized Fatty Acid Content 25-28 vs. 28-32 Weeks Gestation.

Mean normalized fatty acid content comparing 25-28 weeks gestation to 28-32 weeks are reported in Figures 1-4. There were no significant differences in normalized EET, DHET, and HETE content between 25-28 weeks gestation and 28-32 weeks gestation.

Figure 1.

Figure 1.

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Mean normalized EET acid content in 25-28 weeks gestation, 28-32 weeks gestation and postpartum. Blue bars depict 25-28 weeks gestation, orange bars 28-32 weeks gestation, and gray bars ≥ 3 months postpartum with each woman serving as her own control. Error bars represent standard deviations and p value < 0.05 were considered significant.

Figure 4.

Figure 4.

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Mean normalized HETE acid content in 25-28 weeks gestation, 28-32 weeks gestation and postpartum. Nineteen and 20 HETE are not shown as they were below detection quantification limits. Blue bars depict 25-28 weeks gestation, orange bars 28-32 weeks gestation, and gray bars ≥ 3 months postpartum with each woman serving as her own control. Error bars represent standard deviations and p value < 0.05 were considered significant.

Normalized Fatty Acid Content 25-28 Weeks Gestation vs. Postpartum.

Mean normalized fatty acid content comparing 25-28 weeks gestation to postpartum are reported in Figures 1-4 Although trans 8,9-EET showed a trend towards a lower value during 25-28 weeks gestation vs. postpartum (0.55 ± 0.22 vs. 0.64 ± 0.19, p=0.08), no significant changes were observed in any of the epoxyeicosatrienoic (5,6-EET, trans 5,6-EET, 8,9-EET, trans 8,9-EET, 11,12-EET, trans 11,12-EET, 14,15-EET and trans 14,15-EET), dihydroxyeicosatrienoic (5,6-DHET, 8,9-DHET, 11,12-DHET and 14,15-DHET), or hydroxyeicosatetraenoic acids (5-HETE, 8-HETE, 9-HETE, 11-HETE, 12-HETE, 15-HETE, 19-HETE and 20-HETE) when comparing 25-28 weeks gestation to postpartum.

Normalized Fatty Acid Content 28-32 Weeks Gestation vs. Postpartum.

Mean normalized fatty acid content comparing 28-32 weeks gestation to postpartum are reported in Figures 1-4. For the epoxyeicosatrienoic acids, trans 8,9-EET was significantly lower during the late pregnancy window (28-32 weeks gestation) compared to postpartum (0.49 ± 0.18 vs. 0.64 ± 0.19, p=0.01). All other epoxyeicosatrienoic acids were not significantly altered by pregnancy. In contrast, for the dihydroxyeicosatrienoic acids, 8, 9-DHET (1.51 ± 0.46 vs. 1.33 ± 0.29, p<0.05), 11,12-DHET (1.27 ± 0.47 vs. 1.09 ± 0.24, p<0.05) and 14,15-DHET (0.91 ± 0.37 vs 0.74 ± 0.15, p=0.03) were all significantly higher during the late pregnancy window (28-32 weeks gestation) compared to postpartum. All other dihydroxyeicosatrienoic acids were not significantly altered by pregnancy. None of the hydroxyeicosatetraenoic acids were significantly altered by normal pregnancy.

Discussion

An abbreviated AA cascade is depicted in Figure 5. Arachidonic acid, a component of membrane phospholipids, is hydrolyzed from the membrane by phospholipase A2 (PLA2) in response to a number of stimuli. The oxidation of AA via a variety of enzymes (lipooxygenases, cyclooxygenases, CYP4A, CYP4F, CYP2J2 and CYP2C) produces a number of biologically active compounds such as the prostaglandins, thromboxanes, prostacyclins, leukotrienes, and a number of eicosanoids including EETs and HETEs [15]. The EETs are further hydrolyzed by soluble epoxide hydrolase to the DHETs and then eliminated via the kidneys or conjugated with glutathione prior to excretion [17]. EETs can also be re-esterified via acyl CoA dependent mechanisms and can return to the phospholipid membrane. There is a good correlation between plasma and RBC membrane epoxyeicosatrienoic, dihydroxyeicosatrienoic, and hydroxyeicosatetraenoic acids [9]. Both plasma and erythrocyte membranes have been used to measure eicosanoids. Diet has been shown to affect plasma concentrations but has not been shown to affect the pool of DHETs, EETs, and HETE storage in the RBC membrane [16]. Therefore, erythrocyte membrane levels might serve as a better predictor of changes due to pregnancy [15,16].

Figure 5. Effects of late pregnancy on the Arachidonic Acid Cascade.

Figure 5.

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This figure depicts the release of arachidonic acid from the phospholipid membrane, followed by the various pathways for AA metabolism. Lipoxygenases are involved in the formation of the hydroxyeicosatetraenoic acids. Cyclooxygenases are involved in the formation of the prostaglandins and thromboxanes. Reactive oxygen species are involved in the formation of the trans-epoxyeicosatrienoic acids. CytochromeP450 enzymes are involved in the formation of the cis-epoxyeicosatrienoic acids and two of the hydroxyeicosatetraenoic acids. The cis-epoxyeicosatrienoic acids undergo further metabolism by soluble epoxide hydrolase to the dihydroxyeicosatrienoic acids, which are either excreted in the urine or undergo conjugation with glutathione prior to excretion. The cis-epoxyeicosatrienoic acids can also undergo re-esterification via acyl CoA and return to the phospholipid membrane. The green arrows indicate the compounds in which a statistically significant differences (p < 0.05) were observed between 28 to 32 weeks gestation and greater than 3 months postpartum in this study. The direction of the arrow specifies if this was a statistically significant increase or decrease.

There is a great deal of interest in the epoxyeicosatrienoic acids (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) due to their involvement in modulating vascular tone and anti-inflammatory properties [18]. Their corresponding dihydroxyeicosatrienoic acid products (5,6-DHET, 8,9-DHET, 11,12-DHET, and 14,15-DHET) are thought to have unique functions apart from being less active biproducts of EET metabolism. For example, 14,15 dihydroxyeicosatrienoic acid (14,15-DHET) is a potent activator of perioxisome proliferator-activated receptor-alpha, which reduces triglycerides, regulates energy homeostasis, and reduces pain and inflammation. [19, 20]. The 8,9-DHET plays an important role in the autocrine and paracrine systems, having effects on vascular tone, renal tubular transport, cardiac contractility, and inflammation. In addition, 11,12-DHET has arterial smooth muscle relaxation properties [21]. With respect to hydroxyeicosatetraenoic acids, 12-HETE appears to be involved in platelet function, thrombogenesis, angiogenesis, the release of proangiogenic molecule VEGF and mediating proinflammatory and proapoptotic effects of cytokines involved in diabetes. Lastly, 15-HETE is produced in high amounts by airway epithelial cells, eosinophils and reticulocytes, but its physiological effects are still unclear [22]. With the EETs, DHETs, and HETEs playing important roles in human physiology, establishing the normal changes that occur during pregnancy is critical to future elucidation of their roles in pregnancy complications. Thus, the primary objective of this study was to establish the effects of normal pregnancy on RBC membrane EETs, DHETs, and HETEs.

Changes in RBC membrane content of EETs, DHETs, and HETEs during pregnancy can occur through changes in their formation or their elimination. Several cytochrome P450 isoforms are known to be altered during pregnancy. For example, CYP3A, CYP2D6, CYP2C9 and CYP2J2 activities increase during normal pregnancy, whereas CYP1A2 activity is lower during pregnancy. CYP3A, CYP2D6, CYP2C9, and CYP1A2 have been studied in human pregnancies, while CYP2J has only been studied in pregnant rats. [7, 23-26]. Previous work by Catella et al. reported a significant increase in urinary elimination of 8,9 DHET and 11,12 DHET during pregnancy [10]. However, the change in urinary excretion of 14,15 DHET was not statistically different during pregnancy in this small study (7 subjects and age matched controls). Additionally, 5,6 DHET was not among the compounds [10].

The higher CYP2J2 and CYP2C9 activities during pregnancy are expected to increase the formation of 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET. However, we did not detect a significant change in RBC membrane circulating levels for these compounds during pregnancy. Soluble epoxide hydrolase is responsible for the hydrolysis of the EETs and formation of the DHETs. Since 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET contents were not significantly altered by pregnancy, it is likely that soluble epoxide hydrolase activity also increased, leading to the higher content of 8,9-DHET, 11,12-DHET and 14,15-DHET but not 5,6-DHET observed in this study during pregnancy (Figures 1-5). Furthermore, glutathione S-transferase (GST), an enzyme involved in detoxification and clearance of EETs and DHETs, has been reported to have decreased activity during pregnancy. Decreased activity of GST could contribute to a higher concentration of DHET content during pregnancy [27]. To some extent this would have been balanced by the increase in renal filtration occurring during pregnancy and consistent with the increased creatinine clearances seen in this study, which would be expected to lower DHET content [28]. The previous data reporting higher amounts of 8,9 DHET and 11,12 DHET in urine during pregnancy is consistent with their higher RBC content during pregnancy [10].

Previous work evaluating the effects of healthy pregnancy on EETs, HETEs, and DHETs have been limited by methodological challenges. Jiang et al. conducted a study measuring fatty acids with a similar approach to ours utilizing a phospholipid assay, although they had a separate control group rather than each individual serving as their own control. Jiang et al. did not find any difference in RBC total DHETs but did observe an increase in urinary excretion of DHETs [8]. In contrast, we observed significantly higher red blood cell 8,9 DHET, 11,12 DHET and 14,15 DHET during pregnancy. Long et al. found increased concentration of total serum DHET, but no change in 8,9-EET, 11,12-EET, 5-HETE, 8-HETE, 12-HETE and 15-HETE in their unpaired study [9].

Research on trans-EETs and their role in physiological processes is lacking. Trans-EET products are formed by free radical oxidation and are stored in the RBC membrane (Figure 5) [29, 30]. Trans-EETs are cleared similarly to cis-EETs in that they are hydrolyzed to trans-EETs by sEH and then undergo urinary excretion or conjugation with glutathione for excretion [29]. In normal pregnancy, some level of oxidative stress is required to maintain a healthy environment for the fetus, and this level of oxidative stress is much higher in pregnancy compared to the non-pregnant state. We expect that there would be higher levels of trans-EETs in pregnancy due to increased oxidative stress [31]. However, in our study, membrane trans 8,9 EET had lower content during pregnancy. Jiang et al. reported preferential hydrolysis of trans-EET products by sEH compared to cis-EET products [30]. These studies support our findings of reduced trans 8,9-EET during pregnancy, possibly due to increased sEH activity [29,30]. Interestingly, Jiang et al. observed that decreased levels of trans-EETs in rats correlated with oxidative stress and higher blood pressure readings [30].

Noteworthy strengths of the present study include subjects serving as their own control rather than using age-matched controls. To our knowledge, the present study is the only study of its kind in which each subject served as their own control to limit variability. Furthermore, while previous studies have measured EETs, HETEs, and DHETs in plasma or urine, the present study measured concentrations in RBCs membranes and allows for a more accurate representation of EET, HETE, and DHET that are not as influenced by diet [15,16].

Limitations of this study include the lack of mechanistic studies conducted to validate our proposed mechanism for changes in this study, which included increased sEH activity, decreased glutathione conjugation, and increases in CYP2J2 and CYP2C. Although we did not measure the activities of these enzymes, our proposed mechanisms are supported by previously published data [7, 23-27,29,30]. In addition, it would have been ideal to have a timepoint prior to pregnancy to serve as our non-pregnant control. However, it is much more challenging to complete a pregnancy study that includes a pre-pregnancy control day as it takes many women a prolonged period of time to get pregnant. Therefore, utilizing ≥ 3 months postpartum as the non-pregnant control was much more feasible and consistent with how most pharmacokinetic studies are done during pregnancy and postpartum [28]. Another limitation in this study is that we did not statistically adjust for multiple comparisons. Given the limited sample size and the number of statistical tests conducted in this pilot study, we cannot rule out that some of the associations we observed were due to chance. The inter-plate variability was higher than ideal. Since we ran all study days for each individual subject on the same plate and each subject served as their own control, the inter-plate variability is less important. Lastly, all but one of the subjects in the study were lactating on their postpartum study day. Although there are studies investigating AA and EETs in breast milk, there are no studies that have examined the effects of lactation on circulating EETs, HETEs and DHETs [32]. We were unable to examine the effect of lactation on fatty acid concentrations, which could confound interpretation of our results.

4. Conclusions

In summary, healthy pregnancy is associated with an increase in 8,9 DHET, 11,12 DHET and 14,15 DHET. This is likely a result of increased sEH activity and decreased glutathione conjugation, the extent of which must exceed the increase in CYP2J2 and CYP2C activity as well as the increase in renal filtration. In addition, the decrease in trans 8,9-EET content likely reflects an increase in sEH activity that must have exceeded the increase in oxidative stress associated with pregnancy.

Figure 2.

Figure 2.

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Mean normalized DHET acid content in 25-28 weeks gestation, 28-32 weeks gestation and postpartum. Blue bars depict 25-28 weeks gestation, orange bars 28-32 weeks gestation, and gray bars ≥ 3 months postpartum with each woman serving as her own control. Error bars represent standard deviations and p value < 0.05 were considered significant. Significant values are indicated with an asterisk.

Figure 3.

Figure 3.

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Mean normalized trans-EET acid content in 25-28 weeks gestation, 28-32 weeks gestation and postpartum. Blue bars depict 25-28 weeks gestation, orange bars 28-32 weeks gestation, and gray bars ≥ 3 months postpartum with each woman serving as her own control. Error bars represent standard deviations and p value < 0.05 were considered significant. Significant values are indicated with an asterisk.

Highlights.

  • Pregnancy is associated with an increase in 8,9-DHET, 11,12-DHET and 14,15-DHET.

  • Pregnancy is associated with a decrease in trans 8,9-EET.

  • Pregnancy did not alter HETE content.

Acknowledgements

This research was supported in part by the National Institute of General Medical Sciences grant # R01GM124264 (M.F.H.), and National Heart, Lung and Blood Institute grant # R01HL128709 (R.A.T.). The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences, the National Heart, Lung and Blood Institute or the National Institutes of Health.

Footnotes

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Conflict of Interest Statement

The authors declare that there are no conflicts of interest.

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