Association of Lipid Peroxidation Product 4-Hydroxynonenal with Post-Traumatic Stress Disorder

Repeated activation of the hypothalamic-pituitary-adrenal axis system, sleep disturbances, and other symptoms related to posttraumatic stress disorder (PTSD) elevate reactive oxygen species, increase inflammation, and accelerate cellular aging, leading to neuroprogression and cognitive decline. However, there is no information about possible involvement of 4-hydroxynonenal (4-HNE), the product of lipid peroxidation associated with stress-associated diseases, in the complex etiology of PTSD. Therefore, the aim of this study was to compare the plasma levels of 4-HNE between war veterans with PTSD (n = 62) and age-, sex- and ethnicity- matched healthy control subjects (n = 58) in order to evaluate the potential of HNE-modified proteins as blood-based biomarker of PTSD. The genuine 4-HNE-Enzyme-Linked Immunosorbent Assay (HNE-ELISA), based on monoclonal antibody specific for HNE-histidine (HNE-His) adducts, was used to determine plasma HNE-protein conjugates. Our results revealed significantly elevated levels of 4-HNE in patients with PTSD. Moreover, the accumulation of plasma 4-HNE seems to increase with aging but in a negative correlation with BMI, showing specific pattern of change for individuals diagnosed with PTSD. These findings suggest that oxidative stress and altered lipid metabolism reflected by increase of 4-HNE might be associated with PTSD. If confirmed with further studies, elevated 4-HNE plasma levels might serve as a potential biomarker of PTSD.


Introduction
Posttraumatic stress disorder (PTSD) is a trauma and stress-related disorder [1] that occurs in a portion of individuals exposed to a traumatic experience or traumatic events. The prevalence of PTSD differs between different countries and between civilian and military population. However, the lifetime prevalence of PTSD in the general population is estimated at about 8% [2]. The prevalence of combat-related PTSD in the United States is approximately 11-20% [3], while the prevalence of PTSD in Croatian war veterans is estimated between 14-40% [4]. PTSD is a serious and often disabling condition comorbid with different psychiatric and somatic comorbidities, such as cardiovascular and pulmonary diseases, metabolic syndrome, increased inflammation, and autoimmune disorders [5].
Subjects were enrolled in the period between autumn of 2015 and spring of 2017. Individuals with combat-related PTSD were diagnosed using the Structured Clinical Interview (SCID) based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria [1]. At the time of sampling, subjects with PTSD were not receiving any psychiatric medication for at least 30 days. Individuals with PTSD were 38-75 years old and experienced war-related trauma during the Homeland War in Croatia (1991)(1992)(1993)(1994)(1995). Healthy control subjects were 38-76 years old and were evaluated with the same diagnostic instruments as PTSD subjects. Enrollment of both groups of subjects followed the same inclusion/exclusion criteria. Exclusion criteria for all subjects were chronic drug abuse, alcohol dependence, diagnosis of depression, schizophrenia, bipolar disorder, adult attention deficit hyperactivity disorder, Alzheimer's disease, current or recent (previous 3 months) alcohol abuse, and/or the use of lipid lowering agents and antihypertensive and antidiabetic medication. All participants were additionally evaluated according to the International Classification of Diseases (ICD-10) to exclude potential somatic diseases, such as fibrosis, sclerosis, cirrhosis, and malignant liver disease (alcoholic liver cirrhosis (K70.3), alcoholic liver fibrosis and sclerosis (K70.2), and hepatocellular carcinoma (C22.0)). The aims and procedures of the study were explained in detail to all participants. Written informed consent was obtained from all subjects and care was taken to ensure that all procedures contributing to this work agree with the ethical standards of the relevant institutional and national human research ethics committees. All procedures were approved by the Ethics Committee of the University Psychiatric Hospital Vrapce, Zagreb, Croatia, and they were consistent with the ethical norms and standards laid down in the Declaration of Helsinki.

Blood Sampling
Blood samples were collected between 7:30 and 8:00 a.m. after overnight fasting, using BD Vacutainer™ glass blood-collection tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with acid citrate dextrose (ACD). Immediately after blood sampling, platelet-poor plasma was separated by series of centrifugation and stored at −80 • C.

Anthropometric and Biochemical Measurements
Biochemical and anthropometric characteristics were determined at the Laboratory of the University Psychiatric Hospital Vrapce. Height was measured to the nearest 0.5 cm. Body weight was measured with a digital scale to the nearest 0.1 kg. BMI was calculated as weight (kg) divided by height (m 2 ).
Lipid levels were determined using commercial tests and Siemens Dimension Xpand analyzer. Total cholesterol (normal values < 5 mmol/L) was determined with cholesterol oxidasephenol aminophenazone method, while triglycerides-TG (normal values < 1.7 mmol/L), lowdensity lipoprotein-LDL (normal values < 3 mmol/L), and high-density lipoprotein-HDL (normal values > 1.2 mmol/L) were determined using the enzymatic colorimetric assay. Fasting glucose level (normal values 4.4-6.4 mmol/L) was determined using hexokinase (Glucose System Reagent 800, Olympus AU640, Olympus America Inc., Center Valley, PA, USA) and was read on a Dimension Xpand Plus Integrated Chemistry System.

4-HNE-ELISA
The level of 4-HNE protein conjugates was determined by an in-house protocol [27,28]. Briefly, the concentration of plasma samples, quantified by the Bradford method [29] and standards ranging from 0 to 250 pmol of 4-HNE-bovine serum albumin (BSA) conjugates/mg of protein, was adjusted to 10 mg/mL. Each standard/diluted plasma sample (10 µL) was added into 100 µL of 0.05 M carbonate binding buffer (pH 9.6; 0.015 M sodium carbonate, 0.035 M sodium bicarbonate) per well of an ELISA plate (Nunc Immuno Maxisorp, Thermo Scientific, Nunc A/S, 4000 Roskilde, Denmark) in triplicate. Thus, the prepared plate was incubated for 5h at 4 • C, washed once with phosphate-buffered saline (PBS; 200 µL), blocked with a blocking solution (5% fat-free dry milk in carbonate binding buffer) for 3h at room temperature (RT), and washed once with washing buffer (0.1% Tween Biomolecules 2021, 11, 1365 4 of 12 20 in PBS). Primary antibody (a generous gift from Dr. Waeg, [30]) (1:100) in 1% BSA in PBS was incubated overnight (ON) at 4 • C. To eliminate sample background values, one well of each sample was incubated with 1% BSA in PBS (without primary antibody). The next day, the plate was incubated with peroxidase blocking solution (3% H 2 O 2 in blocking solution) for 30 min at RT, goat anti-mouse secondary antibody solution in 1% BSA in PBS (1:100; Dako) for 1h at RT and with freshly prepared 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate solution (0.05 mg/mL) for 30 min at RT when the reaction was stopped with 50 µL of stopping solution (2 M sulfuric acid). Absorbance was read at 450 nm, with the reference filter set to 620 nm. Before each step, the plate was washed 5 times with washing buffer. Plasma concentrations of 4-HNE protein conjugates were interpolated from the standard curve and expressed as pmol 4-HNE protein conjugates/mg of proteins.

Statistical Analysis
The results are expressed as medians and 25th (Q1) and 75th (Q3) percentiles and were evaluated with GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA, USA). The normality of the distribution was assessed with the Kolmogorov-Smirnov test. Since most of the analyzed parameters were not normally distributed, non-parametric analyses were used. Multiple linear regression analysis was performed to examine the possible effects of age, body mass index (BMI), diagnosis, fasting glucose, cholesterol, HDL, LDL, TG, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gammaglutamyl transferase (GGT) levels on plasma 4-HNE concentration, using 4-HNE plasma concentration as a dependent variable and other analyzed parameters as independent variables. Differences in the distribution of 4-HNE, anthropometric, and biochemical characteristics between veterans with PTSD and control subjects were evaluated using Mann-Whitney tests, while the correlations were analyzed with Spearman's rank tests. All tests were two-tailed, and α was set at 0.05. G*Power 3 Software was used to determine a priori sample size and actual power. For the Mann-Whitney U test (with α = 0.05; power (1 − β) = 0.80; a medium effect size ω = 0.50), the total desired sample size was 53 per group, and the actual sample size was 58 in healthy controls group and 62 for the group with diagnosed PTSD. Therefore, we had the appropriate sample size and statistical power to detect significant differences in the studied groups.

Results
Anthropometric and biochemical characteristics of healthy control subjects and patients with PTSD are presented in Table 1. There were no differences in age (p = 0.684) and BMI (p = 0.233) between the two groups of subjects. Additionally, levels of cholesterol (p = 0.472), HDL (p = 0.417), LDL (p = 0.975), TG (p = 0.732), AST (p = 0.573), ALT (p = 0.908), and GGT (p = 0.227) were similar between healthy individuals and PTSD subjects. However, PTSD subjects had higher levels of fasting glucose (p = 0.032) when compared to healthy controls ( Table 1).
The significant association of plasma 4-HNE concentration with the diagnosis of PTSD was confirmed by comparing the 4-HNE levels between two groups of subjects ( Figure 1) using the Mann-Whitney U test (U = 1252.0; p = 0.006). Results suggested significantly higher concentration of HNE-protein adducts in PTSD subjects (median = 25.26 pmol/mg protein) when compared to healthy controls (median = 22.43 pmol/mg protein).  with Mann-Whitney U test and shown as median and 25th (Q1) and 75th (Q3) percentiles.    The association between the concentration of 4-HNE-protein adducts in plasma and different anthropometric and biochemical parameters was analyzed by Spearman's rank correlation in both groups of subjects (Table 2). There was no significant correlation (Table 2) between plasma 4-HNE levels and all analyzed biochemical parameters (fasting glucose, cholesterol, HDL, LDL, TG, AST, ALT, GGT). We observed a significant positive correlation between plasma 4-HNE concentration and age (p < 0.001) in healthy control subjects and patients with PTSD (Table 2, Figure 2). A significant negative correlation of 4-HNE plasma levels with BMI (Table 2, Figure 2) was observed only in PTSD subjects (p = 0.002).

Discussion
This study examined the association of 4-HNE concentration as a measure of lipid peroxidation with the development of PTSD by comparing the concentration of 4-HNEprotein adducts in plasma samples of PTSD subjects in relation to healthy controls. Our results revealed significantly elevated levels of 4-HNE in patients with PTSD compared to healthy control subjects and suggested that the accumulation of plasma 4-HNE increases with aging. The results also indicate that plasma 4-HNE levels negatively correlate with BMI but only in individuals diagnosed with PTSD, thus confirming previous findings on the altered lipid metabolism in PTSD since such a trend was not seen in healthy control subjects [10].
It should be mentioned here that accumulation of 4-HNE in blood vessels progresses by aging, reaching its plateau between the age of 60 to 65 years [31]. Accumulation of 4-HNE in arteries, notably in the aorta, is also known to be influenced by fat-rich food and (oxidative) stress [32]. However, the accumulation of 4-HNE in the blood vessels is not necessarily irreversible, indicating that such 4-HNE-protein adducts might not only contribute to pathogenesis of atherosclerosis but could also be the source of systemic vascular stress [31,32]. Furthermore, in obese, diabetic patients, the blood-originating 4-HNE accumulates in adipose tissues and alters growth and metabolism of the (pro)adipocytes [33,34], which might cause inflammation relevant for metabolic syndrome and systemic vascular stress that is not only chronic but also acute. In favor of this assumption are recent findings revealing vascular stress as a possible cause of abundant blood-originating 4-HNE accumulation in the lungs of patients with SARS-CoV-2 infection associated with the lethal outcome of COVID-19 [35].
Studies examining the direct effect of exposure to a traumatic experience or traumatic events on oxidative stress measures in humans are scarce. However, there are indications that psychological stress in childhood and lifetime chronic stress promotes oxidative stress [36,37]. Different blood biomarkers of oxidative stress were found to be elevated in chronically stressed caregivers [38], in college students during examination periods [39,40], and in individuals who lost a spouse or a close relative [41]. Many psychiatric disorders are associated with oxidative stress [42], such as depression with increased oxidative DNA damage [43][44][45] or 4-HNE [46]. Indeed, increased levels of 4-HNE found in coronary artery disease patients with depression and their attenuation after cardiac rehabilitation associated with the improvement of depression symptoms imply its importance in the progression of depression and as a biomarker of depression symptoms [46]. Studies in patients with bipolar disorder and schizophrenia show either significantly higher [47,48] or no difference [49] in 4-HNE levels.
The involvement of oxidative stress with the diagnosis of PTSD was studied using various indicators. Decreased levels of erythrocyte SOD and GPx suggest a poorer response to oxidative stress in patients with PTSD, which could be a consequence of impaired activity and/or synthesis of these two enzymes [11]. Lipid peroxidation byproduct MDA was found to be increased in both the military population with PTSD [50] and civilian earthquake survivors who developed PTSD [12]. Both studies [12,50] suggested higher rates of lipid peroxidation and decreased antioxidant capacity associated with PTSD. Another study did not detect any significant differences in SOD, GPx, and catalase activity between patients with PTSD and healthy subjects; however, they reported a significant positive correlation between SOD activity, GPx activity, and the symptom severity in PTSD patients [51]. Tezcan and colleagues also reported a positive correlation trend between MDA levels and symptom severity [51]. A preliminary study by Michels and colleagues suggested higher levels of γ-aminobutyric acid and glutathione in the dorsolateral prefrontal cortex and anterior cingulate cortex of PTSD subjects [52]. The study that included active soldiers who took part in the Croatian war in the period between 1991 and 1994 found no relationship between oxidative damage markers and the diagnosis of chronic PTSD [15]. No correlation between oxidative stress levels and PTSD was confirmed in individuals who developed PTSD following a sexual trauma, although the results suggested a decrease in both cortisol and 8-hydroxy-2 -deoxyguanosine (8-OHdG) levels with the time that has elapsed since the trauma exposure [53].
The link between PTSD and oxidative stress is still not understood. However, the available evidence emphasizes the importance of chronic and repeated activation and dysregulation of the HPA [54] axis, which is a consequence of re-experiencing the trauma. Abnormal functioning of the HPA axis has long been associated with PTSD pathophysiology and such repeated activation of the HPA system is considered the main cause of stress-related damage to the brain [54]. The model of glucocorticoid-hippocampal atrophy [55] suggests that glucocorticoids, which are released after exposure to stress, execute their neurotoxic effects on specific brain regions, with the hippocampus being extremely sensitive to such action due to the high density of glucocorticoid receptors. The association of increased glucocorticoid levels and oxidative stress was supported by animal studies showing that the administration of glucocorticoids can affect the parameters of oxidative stress [56]. A study by Sato and colleagues showed a significant association between increased hippocampal lipid oxidation, reduced antioxidant enzyme activity, and subcutaneous corticosterone administration in rats [57]. Thus, the evidence from animal studies leads to conclusion that chronic trauma-related HPA-axis activation is a key mechanism of glucocorticoid-related oxidative stress damage and emphasizes the importance of this process in the development of PTSD. Regarding the HPA axis, glucocorticoids affect the expression of proinflammatory cytokines [58,59]. Interaction between PTSD and oxidative stress could also be mediated by the sleep disturbances, very common symptoms of PTSD. Normal sleep is necessary for restoring oxidant/antioxidant balance and ensuring metabolic homeostasis in the brain [60]. The link between sleep deprivation and oxidative stress was confirmed in the case of insomnia, demonstrating a significant increase in MDA levels and a reduction of GPx activity in subjects with insomnia compared to controls [61]. Animal studies have further contributed to elucidating the link between oxidative stress and sleep deprivation. They have suggested that the lack of sleep can lead to hippocampal oxidative stress and cause memory deficits in mice; the effect can be blocked by antioxidant agents, such as melatonin, N-tert-butyl-alpha-phenylnitrone, or vitamin E [62]. Sleep deprivation was also found to increase the expression of different proinflammatory molecules, such as C-reactive protein, tumor necrosis factor-α, and interleukins [63,64]. All these studies suggest that oxidative stress and inflammation could be the main mechanisms contributing to accelerated aging, cognitive decline, and neurodegeneration as consequences of chronic PTSD.
Our study points to the same direction with a limitation of the study involving only male subjects since the PTSD group included only male combat-exposed war veterans, while 4-HNE levels were determined only at one-time point (yet almost 30 years after the traumatic events of the war). Therefore, in further studies, civilian trauma victims and female subjects should be included. Although this study had strict exclusion criteria, one of the limitations is that we did not take into account the possibility of imbalanced nutrition state. On the other hand, the strengths of the study are in the evaluation of various factors that might affect 4-HNE levels, such as fasting glucose, AST, ALT, GGT, cholesterol, HDL, LDL, and TG. To the best of our knowledge, this is the first study that evaluated 4-HNE-protein levels in PTSD. While including subjects in the study, different comorbidities were taken into account since they often accompany the diagnosis of PTSD and are known to contribute to the elevation of general somatic oxidation state. The subjects with chronic drug abuse, alcohol dependence, diagnosis of different psychiatric disorders, and/or the use of lipid lowering agents and antihypertensive and antidiabetic medication were excluded. We also excluded potential somatic diseases, such as fibrosis, sclerosis, cirrhosis, and malignant liver disease. The included subjects comprised ethnically homogenous male populations matched and controlled for biochemical measures that might affect 4-HNE levels.

Conclusions
Our results revealed significantly elevated levels of 4-HNE in patients with PTSD. The accumulation of plasma 4-HNE seems to increase both in healthy people and in PTSD patients with aging but in a negative correlation with BMI, showing specific pattern of change for individuals diagnosed with PTSD. These findings suggest that oxidative stress and altered lipid metabolism reflected by increase of 4-HNE might be associated with PTSD.
In conclusion, since 4-HNE is already considered as important clinical biomarker of numerous stress-and age-associated diseases [65][66][67], we believe that further studies will prove its importance as a pathophysiological factor and biomarker for PTSD.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in the study are available on request from the corresponding author (N.Ž.) and N.P. The data are not publicly available due to privacy/ethical restrictions, since they contain information that could compromise the privacy of research participants.