Relationship Between 8-iso-prostaglandin-F2α and Predicted 10-Year Cardiovascular Risk in Hypertensive Patients

Academic Editor: Panagiotis Georgianos Received: 1 February 2025 Revised: 25 February 2025 Accepted: 27 February 2025 Published: 4 March 2025 Citation: Geraci, G.; Sorce, A.; Zanoli, L.; Cuttone, G.; Calabrese, V.; Pallotti, F.; Paternò, V.; Ferrara, P.; Dominguez, L.J.; Polosa, R.; et al. Relationship Between 8-iso-prostaglandin-F2αand Predicted 10-Year Cardiovascular Risk in Hypertensive Patients. Life 2025,15, 401. https://doi.org/10.3390/ life15030401 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Relationship Between 8-iso-prostaglandin-F2αand Predicted 10-Year Cardiovascular Risk in Hypertensive Patients Giulio Geraci 1, Alessandra Sorce 2, Luca Zanoli 3, Giuseppe Cuttone 1, Vincenzo Calabrese 1, Francesco Pallotti 1, Valentina Paternò4, Pietro Ferrara 5,6 , Ligia J. Dominguez 1, Riccardo Polosa 1,7 , Jacob George 8, Giuseppe Mulè2and Caterina Carollo 2,* 1Department of Medicine and Surgery, Kore University of Enna, 94100 Enna, Italy; [email protected] (G.G.); [email protected] (G.C.); vincenzo.calabr[email protected] (V.C.); [email protected] (F.P.); [email protected] (L.J.D.); [email protected] (R.P.) 2Unit of Nephrology and Dialysis, Hypertension Excellence Centre, Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties (PROMISE), University of Palermo, 90133 Palermo, Italy; [email protected] (A.S.); [email protected] (G.M.) 3 Nephrology, Department of Clinical and Experimental Medicine, University of Catania, 95124 Catania, Italy; [email protected] 4Operative Unit of Diabetology, Umberto I Hospital, Provincial Health Authority (ASP) of Enna, 94100 Enna, Italy 5 Center for Public Health Research, University of Milan–Bicocca, 20900 Monza, Italy; pietr[email protected] 6Laboratory of Public Health, IRCCS Istituto Auxologico Italiano, 20149 Milan, Italy 7Center of Excellence for the Acceleration of Harm Reduction, University of Catania, 95124 Catania, Italy 8Cardiovascular Medicine and Therapeutics, University of Dundee Medical School, Ninewells Hospital, Dundee DD1 9SY, UK; [email protected] *Correspondence: caterina.car[email protected] Abstract: Background: 8-iso-prostaglandin-F 2α (8-iso-PGF 2α ) is a recognized marker of oxidative stress. Previous studies suggested that 8-iso-PGF 2α plays an important role in the pathogenesis of hypertension and cardiovascular (CV) diseases. However, limited data exist on the prognostic role of 8-iso-PGF 2α in hypertensive patients undergoing primary prevention. The aim of this study was to assess the relationship between 8-iso-PGF 2α and 10-year CV risk, as predicted by validated equations in hypertension patients without CV diseases. Materials and methods: A total of 432 individuals aged 40–75 years were enrolled. Plasma 8-iso-PGF 2α was assessed through the ELISA method. CV risk was calculated by using the Framingham Risk Score (Fr-S) and the Atherosclerosis Cardiovascular Disease Risk Score (ASCVD-S). Low, moderate, or high CV risks were defined according to validated cutoffs. Results: Individuals with higher CV risk had significantly greater 8-iso-PGF 2α values compared to those with low or moderate CV risk (p< 0.001). 8-iso-PGF 2α correlated strongly with Fr-S and ASCVD-S in the entire population and in patients with normal renal function (all p< 0.001) but not in patients with eGFR < 60 mL/min/1.73 m 2 . These associations remained significant after adjustment for traditional factors included in the CV risk equations in the overall population and in patients with normal renal function. The 8-iso-PGF 2α cutoffs that best distinguished patients with high CV risk were 310 pg/mL for Fr-S and 264 pg/mL for ASCVD-S in the overall population, with significant differences between the groups divided by eGFR (all p< 0.001). Conclusions: These findings highlight the potential utility of 8-iso-PGF 2α as a biomarker for refining cardiovascular risk stratification in hypertensive patients, particularly those with preserved renal function. Future studies should explore its prognostic value in longitudinal cohorts and assess its integration into clinical risk models to enhance early prevention strategies for cardiovascular disease. Keywords: oxidative stress; 8-iso-prostaglandin-F 2α cardiovascular risk; hypertension; chronic kidney disease; inflammation; prevention; prognosis Life 2025,15, 401 https://doi.org/10.3390/life15030401 Life 2025,15, 401 2 of 14 1. Introduction The 8-iso-prostaglandin-F 2α (8-iso-PGF 2α ) is a bioactive compound mainly formed in humans via the free radical-mediated peroxidation of arachidonic acid in membrane phospholipids [ 1 ]. It serves as a valuable biomarker of in vivo lipid oxidation and a surrogate indicator of increased reactive oxygen species production and reduced nitric oxide bioavailability. As such, it is considered a sensitive and specific index of oxidative stress [ 2 , 3 ]. Previous studies have demonstrated that 8-iso-PGF 2α induces vasoconstriction, platelet activation, and smooth muscle proliferation in blood vessels [ 4 ]. These effects contribute to the impairment of endothelium-mediated vasodilatation and promote a pro-thrombotic and pro-inflammatory state [3–6]. There is considerable evidence that F 2 -isoprostanes are involved in the pathogenesis of hypertension and atherosclerosis, and their important role in the development of cardiovascular diseases has also been described [ 7 – 10 ]. Oxidative stress contributes to the adverse effects of cardiovascular risk factor by inducing endothelial dysfunction and morphofunctional changes in microcirculation, both of which are strong predictors of organ damage and cardiovascular outcomes [5,6,11,12]. Over the years, several mathematical models have been developed to estimate global risk of cardiovascular events by integrating the partial information of each major risk factor. Among these, the Framingham Risk Score (Fr-S) and the Atherosclerosis Cardiovascular Disease Risk Score (ASCVD-S) have become widely adopted due to their simplicity and validation across diverse populations [ 13 – 15 ]. Developed from longitudinal trial data, these scores can estimate the 10-year risk of cardiovascular diseases, and their use is recommended to assess overall cardiovascular risk, providing a comprehensive approach to managing hypertensive patients [15–17]. The early detection of cardiovascular risk in hypertensive patients is crucial for implementing timely interventions that can reduce the progression of cardiovascular disease and associated complications. Identifying high-risk individuals at an early stage allows for targeted lifestyle modifications, optimized pharmacological treatment, and personalized preventive strategies. The use of reliable biomarkers, such as 8-iso-PGF 2α , as a marker of in vivo oxidative stress, can enhance risk stratification by providing objective measures of oxidative stress, a key contributor to vascular dysfunction [ 1 ]. Integrating such biomarkers into routine clinical assessments may improve risk prediction, facilitate early therapeutic decisions, and ultimately contribute to the prevention of hypertension-related cardiovascular events [7–9,18]. In this context, 8-iso-PGF 2α emerges as a potential mediator between oxidative stress and cardiovascular risk assessment, potentially serving as a predictor of cardiocerebrovascular diseases across diverse populations [ 7 – 9 ]. While some studies support this association, conflicting data remain [ 18 ]. For example, a systematic review by Zhang et al., summarizing findings from 20 studies, reported a significant correlation between elevated F 2 -isoprostanes levels in urine or blood and cardiovascular disease [ 9 ]. However, the authors emphasized the need for further research to clarify the role of F2-isoprostanes as a non-specific indicator of cardiovascular disease. Given the limited evidence on the prognostic value of 8-iso-PGF 2α , a specific F 2 - isoprostane, in hypertensive patients, particularly in the context of primary prevention, our study seeks to investigate its potential as a biomarker for predicting 10-year cardiovascular risk. Importantly, no prior research has explored the relationship between oxidative stress, as measured by 8-iso-PGF 2α , and cardiovascular risk predictions derived from validated risk models in hypertensive patients. Addressing this gap, our study aims to provide new insights into the clinical relevance of oxidative stress markers in cardiovascular risk stratification. Life 2025,15, 401 3 of 14 2. Materials and Methods 2.1. Study Design and Population This cross-sectional observational design was performed on 432 essential hypertensive patients selected from Caucasian patients consecutively attending the Nephrology and Hypertension Section of the University Hospital of Palermo, Italy, for specialist advice, between January 2024 and October 2024. Patients meeting the following criteria were excluded from this research (see Supplementary Figure S1): − Aged <40 and >75 years old, to align with the application range of Fr-S and ASCVD-S; − Renovascular, endocrine, or malignant hypertension or hypertension associated with obstructive sleep apnea syndrome, as described in detail in previous studies [19,20]; −Renal replacement therapy (transplanted or dialysis patients); − Pharmacological treatment for cardiac rhythm or conduction abnormalities, in order to minimize potential confounders; − Use of nonsteroidal or steroidal anti-inflammatory medications within 4 weeks before the start of the study. − History of cerebrovascular disease, coronary heart disease, or symptomatic peripheral arterial disease; −Hospitalization for CV cause in the previous 6 months; − Major non-cardiovascular diseases (history of liver cirrhosis, chronic obstructive lung disease, or neoplasms). The study protocol conformed to the ethical guidelines of the Declaration of Helsinki on ethical principles for medical research involving human subjects (REF) and was approved by the Local Review Board. Written informed consent was obtained from each patient. 2.2. Clinical and Laboratory Evaluation Careful clinical history and physical examination were performed in all patients. Individuals who reported smoking cigarettes regularly during the past year were considered current smokers. Body weight and height were measured by a nurse, and body mass index (BMI) was calculated as body weight divided by squared height (kg/m 2 ). Patients with a history of diabetes (or on treatment with antidiabetic drugs) or with fasting serum glucose levels of ≥ 126 mg/dL were considered diabetics. For individuals with fasting serum glucose levels of between 100 and 125 mg/dL, the diagnosis of diabetes was confirmed based on either glycated hemoglobin or 2 h plasma glucose during an oral glucose tolerance test. Clinic blood pressure (BP) was recorded by a doctor as the mean of three consecutive measurements obtained at 2 min intervals using a validated electronic oscillometric device (WatchBP Office, Microlife AG, Widnau, Switzerland), after 5 min of rest in a sitting position. According to the 2023 European Society of Hypertension/European Society of Cardiology guidelines, hypertension was defined as a BP ≥ 140/90 mmHg or treatment with antihypertensive drugs [17]. At 08:30 h on the day of the study, the overnight-fasted patients were placed in a supine position, and blood samples were obtained from an indwelling forearm venous catheter. Routine biochemical parameter determination was performed in all patients with standard techniques using an autoanalyzer (Boehringer Mannheim for Hitachi system 911, Mannheim, Germany). Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. Estimated glomerular filtration rate (eGFR) was determined using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. The 8-iso-PGF 2α was measured by a solid-phase, specific enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Assay Design Inc., Ann Arbor, MI, USA), with particular attention to minimizing interference from other serum components. After centrifugation at 4 ◦ C, blood samples were frozen at − 80 ◦ C and processed within 2 months of collection. Life 2025,15, 401 4 of 14 According to the manufacturer’s recommendations, the test sensitivity was 16.3 pg/mL, and the inter-assay coefficient of variation was <9%. High-sensitivity C-reactive protein (CRP) was also measured using a commercially available ELISA kit (Diagnostic Biochem, London, Ontario, Canada), with a sensitivity of 10 ng/mL, an inter-assay coefficient of variation of <10%, and an intra-assay coefficient of variation of <8%. Cardiovascular risk score: Past medical history, clinical data, and laboratory tests were collected in all patients to predict the 10-year risk of cardiovascular events using validated equations of Fr-S and ASCVD-S. These mathematical sexand race-specific models provide an estimate of overall 10-year cardiovascular risk, and were, respectively, derived by the Framingham cohort study and pooled cohorts of participants from several large studies, including the Atherosclerosis Risk in Communities (ARIC) study, the Cardiovascular Health Study, and the Coronary Artery Risk Development in Young Adults (CARDIA) study. Based on specific cut-offs, the patients were classified into low CV risk (Fr-S < 10%; ASCVD < 7.5%), intermediate CV risk (Fr-S ≥ 10% and <20%; ASCVD ≥ 7.5% and <15%), or high CV risk (Fr-S ≥20%; ASCVD ≥15%) [15–21]. 2.3. Statistical Analysis Statistical analyses were performed using the IBM SPSS Statistics software package, version 23, for Macintosh (SPSS, Chicago, IL, USA). Statistical analysis was initially performed on the entire study population. Given the well-established link between 8-isoPGF 2α and kidney function, as noted in previous studies [ 22 , 23 ], statistical analysis was subsequently conducted on two subgroups based on eGFR: ≥ 60 mL/min/1.73 m 2 (n= 279) and <60 mL/min/1.73 m 2 (n= 153). For further analyses, the population was divided into three groups according to validated 10-year cardiovascular risk cutoff values (10% and 20% for Fr-S; 7.5% and 15% for ASCVD-S) [15–21]. The normal distribution of continuous variables was assessed using the Kolmogorov– Smirnov test. Continuous variables were reported as means ± standard deviation (SD). Triglycerides, Fr-S, and ASCVD-S, which had skewed distributions, were log-transformed to satisfy distributional assumptions before applying parametric tests. These variables were presented as median and interquartile range (IQR). Categorical variables were expressed as percentages. Comparisons of continuous variables between groups were conducted using Student’s t-test for unpaired data or analysis of variance (ANOVA) with the Holm–Sidak test for multiple comparisons, as appropriate. For the categorical variables, comparisons were performed using the χ2 test, with the Monte Carlo method employed to compute exact two-tailed α-values. Univariate regression analyses with Pearson’s correlation coefficients were used to examine the relationships between 8-iso-PGF 2α with Fr-S, ASCVD-S, and the other variables. Stepwise multivariate regression analyses were performed with Fr-S (or alternatively ASCVD-S) as the outcome variable. Covariates included: age, sex (0 = females; 1 = males), diabetes (0 = no; 1 = yes), current smoking habit (0 = no; 1 = yes), antihypertensive therapy (0 = no; 1 = yes), BMI, serum total cholesterol, HDL and LDL-cholesterol, clinic systolic BP, eGFR, and 8-iso-PGF 2α . To further assess the influence of renal function, treated as continuous variable (eGFR), on the relationship between Fr-S (or alternatively ASCVD-S) and 8-iso-PGF 2α , additional multivariate models were analyzed in the population divided into two groups based on eGFR values ( ≥ 60 mL/min/1.73 m 2 or <60 mL/min/1.73 m 2 ). A backward stepwise procedure was used in all analyses, with α equal to 0.15 as the cutoff for variable entry or removal. Collinearity was assessed by calculating the variance inflation factor (VIF): variables with VIF ≥ 2 were excluded from the models. The null hypothesis was rejected with a two-tailed p-value ≤0.05. Life 2025,15, 401 5 of 14 Receiver-operating characteristic (ROC) curves were built for the entire population and for the two groups divided by eGFR to evaluate the accuracy of 8-iso-PGF 2α in detecting a 10-year risk of cardiovascular disease ≥ 20% with Fr-S or ≥ 15% with ASCVD-S. The significance of differences between ROC curves was assessed using the Hanley and McNeil method. The null hypothesis was rejected at a p-value of ≤0.05. 3. Results A total of 432 hypertensive patients were enrolled. The mean age of the overall study population was 60 ± 10 years; 59.0% were male and 35.4% had an eGFR of <60 mL/min/1.73 m 2 . Table 1presents the characteristics of the overall study population and the two groups divided by eGFR. Table 1. 8-iso-PGF 2α in patients with low, moderate, or high cardiovascular risk calculated by the Framingham Risk Score or ASCVD Risk Score. Variable * Overall Population (n= 432) eGFR ≥60 (n= 279) eGFR < 60 (n= 153) p-Value ˆ Age (years) 60 ±10 57 ±10 65 ±8<0.001 Male sex, n(%) 255 (59) 173 (64.0) 82 (53.6) NS Smoking habit, n(%) 109 (25.3) 59 (21.15) 50 (32.8) NS Diabetes, n(%) 111 (25.7) 63 (22.6) 48 (31.4) NS Antihypertensive therapy, n(%) 415 (96.1) 270 (96.8) 145 (94.8) NS Clinic systolic BP (mmHg) 142 ±21 143 ±21 140 ±20 NS Clinic diastolic BP (mmHg) 84 ±13 86±14 80 ±11 <0.001 Clinic mean BP (mmHg) 103 ±14 105 ±15 100 ±12 0.001 Clinic pulse pressure (mmHg) 58 ±16 57 ±15 60 ±18 NS Clinic heart rate (bpm) 73 ±10 73 ±10 72 ±11 NS Biochemical parameters Serum glucose (mg/dL) 110.1 ±36.1 108.6 ±31.7 112.8 ±42.9 NS Serum uric acid (mg/dL) 6.43 ±1.65 6.39 ±1.70 6.48 ±1.59 <0.001 Serum total cholesterol (mg/dL) 191.5 ±43.6 193.6 ±40.3 187.6 ±48.9 NS LDL-c (mg/dL) 119.06 ±38.80 121.69 ±37.65 114.27 ±40.32 NS HDL-c (mg/dL) 46.11 ±12.44 47.22 ±11.79 44.10 ±13.35 <0.05 Serum triglycerides (mg/dL) 118 (86–161) 105 (81–152) 136 (104–177) <0.001 Serum creatinine (mg/dL) 1.43 ±1.14 0.92 ±0.16 2.36 ±1.53 <0.001 eGFR (ml/min/1.73 m2)65.9 ±27.5 83.5 ±12.8 33.8 ±16.1 <0.001 Serum sodium (mEq/L) 139 ±3 140 ±3 139 ±3NS Serum potassium (mEq/L) 4.35 ±0.40 4.33 ±0.38 4.37 ±0.43 NS Endothelial disfunctions and cardiovascular risk 8-iso-PGF2α(pg/mL) 292.6 ±125.7 247.2 ±104.7 375.4 ±118.7 <0.001 CRP (mg/dL) 2.40 (1.60–3.30) 2.00 (1.39–2.70) 3.17 (2.40–3.80) <0.001 Framingham Risk Score (%) 7.46 (4.17–14.06) 6.49 (3.60–11.76) 9.44 (6.00–17.83) 0.001 Framingham Risk Score < 10%, n (%) 272 (63.0) 193 (69.2) 79 (51.6) <0.001 Framingham Risk Score ≥20%, n(%) 61 (14.1) 36 (12.9) 25 (16.3) NS ASCVD Risk Score (%) 10.92 (4.92–21.43) 8.25 (4.24–17.28) 15.83 (9.59–28.27) <0.001 ASCVD Risk Score < 7.5%, n(%) 157 (36.3) 129 (46.2) 28 (18.3) <0.001 ASCVD Risk Score ≥15%, n(%) 167 (38.7) 87 (31.2) 80 (52.3) <0.001 * Continuous variables are reported as either mean ± standard deviation or median with interquartile range, based on their distribution. ˆ Comparison between eGFR-based groups; non-significant (NS): p> 0.05. Abbreviations: eGFR—estimated glomerular filtration rate; BP—blood pressure; LDL-c—low-density lipoprotein cholesterol; HDL-c—high-density lipoprotein cholesterol; 8-iso-PGF 2α —8-iso-prostaglandin F 2α ; CRP—C-reactive protein; ASCVD—atherosclerotic cardiovascular disease. Most of the individuals had a low cardiovascular risk, and none had a history of previous cardiovascular events. Patients with low eGFR had significantly higher values of Life 2025,15, 401 6 of 14 8-iso-PGF, Fr-S, and ASCVD-S compared to those with normal eGFR (all p< 0.001), while no significant differences in antihypertensive therapy were observed between groups. Subjects with higher cardiovascular risk had significantly greater values of 8-iso-PGF 2α compared to those with low or moderate cardiovascular risk (Figure 1). Life 2025, 15, x FOR PEER REVIEW 6 of 15 ASCVD Risk Score < 7.5%, n (%) 157 (36.3) 129 (46.2) 28 (18.3) <0.001 ASCVD Risk Score ≥ 15%, n (%) 167 (38.7) 87 (31.2) 80 (52.3) <0.001 * Continuous variables are reported as either mean ± standard deviation or median with interquartile range, based on their distribution. ^ Comparison between eGFR-based groups; nonsignificant (NS): p > 0.05. Abbreviations: eGFR—estimated glomerular filtration rate; BP—blood pressure; LDL-c—low-density lipoprotein cholesterol; HDL-c—high-density lipoprotein cholesterol; 8-iso-PGF2α—8-iso-prostaglandin F2α; CRP—C-reactive protein; ASCVD— atherosclerotic cardiovascular disease. Most of the individuals had a low cardiovascular risk, and none had a history of previous cardiovascular events. Patients with low eGFR had significantly higher values of 8-iso-PGF, Fr-S, and ASCVD-S compared to those with normal eGFR (all p < 0.001), while no significant differences in antihypertensive therapy were observed between groups. Subjects with higher cardiovascular risk had significantly greater values of 8-isoPGF2α compared to those with low or moderate cardiovascular risk (Figure 1). Figure 1. 8-iso-Prostaglandin F2α levels in patients with low, moderate, or high cardiovascular risk (CV) calculated by the Framingham Risk Score or Atherosclerotic Cardiovascular Disease Risk Score. The main univariate correlations of 8-iso-PGF2α, Fr-S, and ASCVD-S in the overall population are presented in Table 2. 257.7 233.3 328.9 292.7 389.3 348.3 200 220 240 260 280 300 320 340 360 380 400 Framingham Risk Score ASCVD Risk Score 8-iso-PGF2 α (pg/ml) ANOVA: p < 0.001 Low CV Risk Moderate CV Risk High CV Risk ANOVA: p < 0.001 Figure 1. 8-iso-Prostaglandin F 2α levels in patients with low, moderate, or high cardiovascular risk (CV) calculated by the Framingham Risk Score or Atherosclerotic Cardiovascular Disease Risk Score. The main univariate correlations of 8-iso-PGF 2α , Fr-S, and ASCVD-S in the overall population are presented in Table 2. Table 2. Main correlations of 8-iso-PGF 2α and cardiovascular risk scores with other variables in the entire study population. 8-Iso-PGF2α Framingham Risk Score ASCVD Risk Score r r r Age (years) 0.383 *** 0.778 *** 0.859 *** Serum glucose (mg/dL) 0.202 *** 0.377 *** 0.345 *** Serum uric acid (mg/dL) −0.051 NS 0.234 *** 0.273 *** Serum total cholesterol (mg/dL) −0.131 ** −0.301 *** −0.160 *** LDL-c (mg/dL) −0.165 *** −0.090 * −0.156 ** HDL-c (mg/dL) −0.027 NS −0.288 *** −0.256 *** Serum triglycerides (mg/dL) 0.090 NS 0.088 NS 0.147 ** Serum creatinine (mg/dL) 0.466 *** 0.127 ** 0.177 *** eGFR (mL/min/1.73 m2)−0.520 *** −0.254 *** −0.338 *** Serum sodium (mEq/L) −0.024 NS −0.085 NS −0.009 NS Serum potassium (mEq/L) 0.086 NS 0.088 NS 0.084 NS Systolic BP (mmHg) 0.188 *** 0.236 *** 0.156 *** Diastolic BP (mmHg) −0.015 NS −0.163 *** −0.247 *** Mean BP (mmHg) 0.083 NS 0.014 NS −0.076 NS Pulse Pressure (mmHg) 0.250 *** 0.430 *** 0.395 *** Heart Rate (bpm) −0.046 NS −0.074 NS −0.094 * CRP (mg/dL) 0.717 *** 0.407 *** 0.404 *** ***: p ≤ 0.001; **: p ≤ 0.01; *: p ≤ 0.05; NS:p> 0.05. Abbreviations: ASCVD—atherosclerotic cardiovascular disease; LDL-c—low-density lipoprotein cholesterol; HDL-c—high-density lipoprotein cholesterol; eGFR—estimated glomerular filtration rate; BP—blood pressure; 8-iso-PGF 2α —8-iso-prostaglandin F 2α ; CRP—C-reactive protein. Life 2025,15, 401 7 of 14 8-iso-PGF 2α was significantly associated with the variables included in the equations used to predict 10-year cardiovascular risk. Furthermore, 8-iso-PGF 2α showed a strong correlation with both Fr-S or ASCVD-S (all p< 0.001) (Figure 2), and these relationships remained significant after adjustment for eGFR values (r = 0.361 and p< 0.001 with Fr-S; r = 0.306 and p< 0.001 with ASCVD-S). When these relationships were assessed separately in the two groups of patients divided by eGFR, 8-iso-PGF 2α was significantly associated with Fr-S and ASCVD-S only in subjects with eGFR ≥ 60 mL/min/1.73 m 2 (respectively r = 0.667 and r = 0.580; all p< 0.001). In contrast, these correlations were not observed in subjects with lower eGFR< 60 mL/min/1.73 m2. Life 2025, 15, x FOR PEER REVIEW 8 of 15 Figure 2. Univariate correlations between8-iso-Prostaglandin F2α levels nd Framingham Risk Score (upper plot) or Atherosclerotic Cardiovascular Disease Risk Score (lower plot) in the entire study population. At the multivariate analyses in the overall population, 8-iso-PGF2α was significantly associated with Fr-S (or alternatively ASCVD-S) independently of other covariates, including eGFR and variables used to calculate the 10-year cardiovascular risk (Table 3). 110100 0 100 200 300 400 500 600 700 800 900 8-iso-PGF2 α (pg/ml) Framingham Risk Score ALL PATIENTS r= 0.474 p < 0.001 0,1 1 10 100 0 100 200 300 400 500 600 700 800 900 8-iso-PGF2 α (pg/ml) ASCVD Risk Score ALL PATIENTS r = 0.465 p < 0.001 0.1 Figure 2. Univariate correlations between8-iso-Prostaglandin F 2α levels nd Framingham Risk Score (upper plot) or Atherosclerotic Cardiovascular Disease Risk Score (lower plot) in the entire study population. Life 2025,15, 401 8 of 14 At the multivariate analyses in the overall population, 8-iso-PGF 2α was significantly associated with Fr-S (or alternatively ASCVD-S) independently of other covariates, including eGFR and variables used to calculate the 10-year cardiovascular risk (Table 3). Table 3. Independent multivariate correlates of Framingham Risk Score [A] and ASCVD Risk Score [B] in the overall study population. [A] Outcome Variable: Framingham Risk Score Regression Coefficients Standardized Bβtp-Value Model (R2= 0.938) Age 0.024 0.683 45.810 <0.001 Diabetes 0.274 0.326 24.988 <0.001 Systolic BP 0.005 0.277 21.928 <0.001 Sex (male) 0.178 0.240 18.354 <0.001 Smoking habit 0.166 0.165 12.780 <0.001 HDL cholesterol 0.002 0.079 5.844 <0.001 Serum total cholesterol 0.001 −0.059 −4.357 0.001 eGFR <0.001 0.066 4.128 0.001 8-iso-PGF2α<0.001 0.052 3.236 0.001 Constant −1.582 - −27.712 <0.001 [B] Outcome Variable: ASCVD Risk Score Regression Coefficients Standardized Bβtp-Value Model (R2= 0.969) Age 0.038 0.891 82.357 <0.001 Diabetes 0.245 0.244 26.431 <0.001 Sex (male) 0.207 0.232 25.019 <0.001 Systolic BP 0.005 0.216 24.131 <0.001 Serum total cholesterol 0.002 0.177 18.455 <0.001 HDL cholesterol −0.006 −0.168 −17.513 <0.001 Smoking habit 0.072 0.060 6.562 <0.001 Antihypertensive therapy 0.129 0.057 6.482 <0.001 eGFR <0.001 0.036 3.150 0.002 8-iso-PGF2α<0.001 0.026 2.285 0.023 Constant −2.384 - −43.679 <0.001 Abbreviations: BP—blood pressure; HDL—high-density lipoprotein cholesterol; eGFR—estimated glomerular filtration rate; 8-iso-PGF 2α —8-iso-prostaglandin F 2α ; ASCVD—atherosclerotic cardiovascular disease. B—unstandardized coefficient, β—standardized regression coefficient, t—t-value, R2—coefficient of determination. Additional multivariate models were constructed for subgroups with eGFR ≥ 60 mL/min/1.73 m 2 and <60 mL/min/1.73 m 2 , and 8-iso-PGF 2α was independently associated with Fr-S and ASCVD-S only in individuals with eGFR ≥60 mL/min/1.73 m2 (all p< 0.001), whereas no significant relationship was observed in individuals with renal impairment. The ROC curves created to assess the global accuracy of 8-iso-PGF 2α in detecting patients with high cardiovascular risk (Fr-S ≥ 20%; ASCVD-S ≥ 15%) are shown in Figure 3. The 8-iso-PGF 2α cutoffs that best distinguished patients with high cardiovascular risk were 310 pg/mL for Fr-S (AUC: 0.767) and 264 pg/mL for ASCVD-S (AUC: 0.718) (Figure 3A,B). Life 2025,15, 401 9 of 14 Life 2025, 15, x FOR PEER REVIEW 10 of 15 (A) (B) Figure 3. Receiver operating characteristic (ROC) curves of 8-iso-Prostaglandin F2α levels for the detection of high cardiovascular risk calculated by Framingham Risk Score or Atherosclerotic Cardiovascular Disease Risk Score in the overall study population (A,B). When ROC curves were compared in patients stratified by eGFR, higher AUC values were observed in patients with eGFR ≥ 60 mL/min/1.73 m2 compared to those with lower eGFR, with significant differences (all p < 0.001). Patients with higher predicted cardiovascular risk had significantly elevated levels of 8-iso-PGF2α, which strongly correlated with the two risk scores considered, particularly in those with normal renal function. The association remained significant after adjusting for traditional risk factors but was not observed in patients with impaired kidney function, suggesting a potential influence of renal status on oxidative stress markers. 4. Discussion A key finding of our study is that 8-iso-PGF2α, a reliable marker of oxidative stress, is independently associated with 10-year cardiovascular risk, as predicted by validated equations in hypertensive patients without overt cardiovascular disease. There is experimental evidence that oxidative stress contributes to the pathogenesis of hypertension [3,11,12], and previous studies have investigated the potential role of 8-isoPGF2α in the process of atherosclerosis and cardiovascular diseases [7–10,22]. Minuz et al. demonstrated the increased urinary excretion of 8-iso-PGF2α in 75 hypertensive individuals compared to 75 pair-matched healthy controls [23], and other authors similarly found elevated urinary F2-isoprastanes in hypertensive patients and individuals at risk for future cardiovascular events [4,10]. Cottone et al. observed higher serum levels of 8-iso-PGF2α in individuals with essential hypertension compared to healthy controls, confirming that oxidative stress is increased in this population [3]. High levels of F2isoprostanes have also be proposed as a biomarker of cardiovascular disease, and the role of 8-iso-PGF2α in cardiovascular events has also been investigated by several authors. In a large general population study, Keaney et al. reported that urinary 8-epi-PGF2α levels were associated with previous cardiovascular diseases [24]: in this study, approximately 13% of participants had a history of prior cardiovascular events, and only one-third of patients had hypertension. In contrast, in our study, none of participants had overt 0 20406080100 0 20 40 60 80 100 Sensitivity 100-Specificity CUT-OFF VALUE 8-iso-PGF2α>310 pg/ml AUC = 0.767 0 20406080100 0 20 40 60 80 100 CUT-OFF VALUE 8-iso-PGF2α> 264 pg/ml AUC = 0.718 Sensitivity 100-Specificity Figure 3. Receiver operating characteristic (ROC) curves of 8-iso-Prostaglandin F 2α levels for the detection of high cardiovascular risk calculated by Framingham Risk Score or Atherosclerotic Cardiovascular Disease Risk Score in the overall study population (A,B). When ROC curves were compared in patients stratified by eGFR, higher AUC values were observed in patients with eGFR ≥ 60 mL/min/1.73 m 2 compared to those with lower eGFR, with significant differences (all p< 0.001; Figure 3A,B). When ROC curves were compared in patients stratified by eGFR, higher AUC values were observed in patients with eGFR ≥ 60 mL/min/1.73 m 2 compared to those with lower eGFR, with significant differences (all p< 0.001). Patients with higher predicted cardiovascular risk had significantly elevated levels of 8-iso-PGF 2α , which strongly correlated with the two risk scores considered, particularly in those with normal renal function. The association remained significant after adjusting for traditional risk factors but was not observed in patients with impaired kidney function, suggesting a potential influence of renal status on oxidative stress markers. 4. Discussion A key finding of our study is that 8-iso-PGF 2α , a reliable marker of oxidative stress, is independently associated with 10-year cardiovascular risk, as predicted by validated equations in hypertensive patients without overt cardiovascular disease. There is experimental evidence that oxidative stress contributes to the pathogenesis of hypertension [ 3 , 11 , 12 ], and previous studies have investigated the potential role of 8-iso-PGF 2α in the process of atherosclerosis and cardiovascular diseases [ 7 – 10 , 22 ]. Minuz et al. demonstrated the increased urinary excretion of 8-iso-PGF 2α in 75 hypertensive individuals compared to 75 pair-matched healthy controls [23], and other authors similarly found elevated urinary F 2 -isoprastanes in hypertensive patients and individuals at risk for future cardiovascular events [ 4 , 10 ]. Cottone et al. observed higher serum levels of 8-iso-PGF 2α in individuals with essential hypertension compared to healthy controls, confirming that oxidative stress is increased in this population [ 3 ]. High levels of F 2 -isoprostanes have also be proposed as a biomarker of cardiovascular disease, and the role of 8-iso-PGF 2α in cardiovascular events has also been investigated by several authors. In a large general population study, Keaney et al. reported that urinary 8-epi-PGF 2α levels were associated with previous cardiovascular diseases [ 24 ]: in this study, approximately 13% of participants had a history