Altered Epoxyeicosatrienoic Acid Signaling in Advanced-Stage Alzheimer’s Disease (Investigation of Epoxyeicosatrienoic Acid Levels in Patients with Advanced Alzheimer’s Disease)

Abstract

The study aimed to compare plasma Epoxyeicosatrienoic Acid (EET) isomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) in patients with advanced-stage late-onset Alzheimer’s Disease (AS-LOAD) versus healthy controls matched by age and sex. 2. Materials and Methods 2.1. Study Population This study included 60 participants: 30 patients with AS-LOAD who were being followed at the Community Mental Health Center of the Nigde Training and Research Hospital, and 30 age- and sex-matched cognitively healthy volunteers recruited from the hospital’s outpatient clinics. The diagnosis of AD in the patient group was made according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V), based on clinical psychiatric evaluation. The control group consisted of individuals without any psychiatric disorders, as confirmed by DSM-V criteria and clinical examination, who volunteered to participate in the study. All procedures were conducted in accordance with the ethical standards of the Declaration of Helsinki. This study was approved by the Nigde Omer Halisdemir University Non-Interventional Clinical Research Ethics Committee (Approval No: 2025/70). 2.2. Collection of Blood Samples Participants and their guardians were informed about the study, and written informed consent was obtained before blood collection. Five mL of blood was collected from each participant into sterile EDTA tubes. The blood samples were centrifuged at 3000 rpm for 10 minutes to separate plasma. Plasma samples were stored at -80°C until EET level analyses were performed. 2.3. Determination of Plasma EET Levels Before the LC-MS/MS experiment, standards for all compounds were purchased from Cayman Chemicals (Ann Arbor, MI). The formic acid, Millipore water, dichloromethane, methanol (MeOH), and ammonium formate used were LC-grade and were obtained from Merck (Darmstadt, Germany). Quantification of plasma EET levels was performed by a validated liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method in the research and development laboratory of Bome Trivitron Industry Products Foreign Trade Inc., as previously described by Duflot et al.[19]. LC-MS/MS analysis was conducted using a Shimadzu 8040 LC-MS/MS system (Shimadzu, Japan) equipped with an electrospray ionization (ESI) source operating in positive ion mode. This system was coupled with a Shimadzu Prominence UFLC system (Shimadzu, Kyoto, Japan), which included a DGU-20A3 degasser, a Nexera-X2 pump, a SIL-20ACHT temperature-controlled autosampler, and a CTO-20AC column oven. Chromatographic separation was performed on a Kinetex C18 column (2.6 μm particle size, 50 mm length × 3 mm inner diameter). Instrument parameters were set to maintain a column temperature of 30°C with a flow rate of 0.6 mL/min. A gradient elution method was employed using methanol/formic acid and ammonium formate/formic acid mixtures as the mobile phases. System control and data acquisition were performed using LabSolutions software, and quantification was conducted in multiple reaction monitoring (MRM) mode. Endogenous EETs were accounted for by normalizing analyte peak areas to zero via linear regression. Calibration curve slopes and linearity were determined from analyte-to-internal standard peak ratios at each spiked concentration. The results were evaluated against the standard calibration curve. 2.4. Statistical Analysis All statistical analyses were performed using IBM SPSS Statistics version 18. The Kolmogorov–Smirnov and Shapiro–Wilk tests were applied to assess the normality of data distribution. Depending on data distribution, comparisons between groups were conducted using the Kruskal–Wallis and Mann–Whitney U tests for nonparametric variables, and one-way ANOVA for demographic parameters such as age and sex. A p-value of ≤ 0.05 was considered statistically significant. In this cross-sectional case-control study, 30 patients with AS-LOAD were compared with 30 cognitively healthy (control) individuals. Plasma EET levels were analyzed by liquid chromatography coupled with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS). The statistical analysis of the demographic characteristics of the AS-LOAD and healthy control groups is presented in Table 1. No statistically significant differences were observed between the control and AS-LOAD groups in terms of mean age or sex distribution (p > 0.05). In our study examining plasma EET levels in patients with AS-LOAD, we found that plasma concentrations of EET isomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) were significantly decreased in AS-LOAD patients compared with age- and sex-matched healthy controls (p < 0.05, Figure 1). Specifically, plasma levels of 14,15-EET (29.37 ± 25.65 vs. 90.80 ± 91.02 ng/dL), 11,12-EET (65.79 ± 38.65 vs. 240.1 ± 237.73 ng/dL), 8,9-EET (57.52 ± 6.46 vs. 144.58 ± 133.77 ng/dL), and 5,6-EET (7.31 ± 8.62 vs. 11.76 ± 11.32 ng/dL) were significantly lower in the patient group compared to the controls (Figure 1). Plasma concentrations of all measured EET regioisomers were significantly lower in patients with AS-LOAD compared with controls. Significant reductions were observed for 14,15-EET (p = 0.007), 11,12-EET (p = 0.001), 8,9-EET (p = 0.007), and 5,6-EET (p = 0.011). EETs are reportedly implicated in AD development, with most previous investigations using animal models and targeted enzymes involved in EET metabolism, particularly soluble epoxide hydrolase (sEH). Furthermore, no studies have specifically examined EET metabolism in patients with advanced AD. This study revealed defective EET signaling with consequent increased sEH activity in AS-LOAD, suggesting that decreased EET might contribute to tau-mediated neurodegeneration, oxidative stress, and neuroinflammation in these patients. This study emphasizes the crucial role of EETs in AD pathology. Targeting EET metabolism could offer a promising therapeutic strategy for preventing AD. Studies that track disease progression are therefore a prerequisite for delaying its disease onset; thus, larger-scale longitudinal studies are needed to confirm these findings.