F 4 -neuroprostanes and F 2 -dihomo-isoprostanes: biomarkers and bioactive oxylipins

– Under condition of oxidative stress, free radical-catalyzed peroxidation of docosahexaenoic acid (DHA) and adrenic acid (AdA) generates in vivo neuroprostanes (NeuroPs) and dihomo-isoprostanes (dihomo-IsoPs), among a large number of key products participating in many pathophysiological processes. These non-enzymatic oxygenated metabolites display a wide range of biological actions (especially DHA-metabolites), and some of them are now considered as the most reliable indicators of oxidative stress in neurogenerative, neurodevelopmental or cardiovascular diseases. In this review, we will present an overview regarding neuroprostanes and dihomo-isoprostanes and discuss about their biological interests.


Introduction
In 1990, Morrow et al. (1990) highlighted the formation in man of the isoprostanes (IsoPs), isomeric to prostaglandins (PGs), by a non-enzymatic mechanism involving the radical peroxidation. These compounds are biosynthetized while arachidonic acid (C20:4 n-6, AA) is linked to the phospholipids. A large number of diastereoisomers, however with major cis-configurations between the lateral chains on the cyclopentane ring, can be generated, as well as several functions on the 5-member ring (diol, enone, hydroxyketone). The metabolites are further released from the membrane by a specific phospholipase A 2 (PLA 2 ) (Stafforini et al., 2006). Later, the same group from the Vanderbilt University described a similar mechanism for the peroxidation of docosahexaenoic acid (C22:6 n-3, DHA), one of the main polyunsaturated fatty acids (PUFAs) in the brain, leading to neuroprostanes (NeuroPs) (Roberts et al., 1998;Nourooz-Zadeh et al., 1998).
It should be known that the lipid peroxidation of PUFAs (here DHA, Fig. 1) begins with the abstraction of an atom of hydrogen by HO . , in one of the 5 bis-allylic positions, either on C-5, or C-8, or C-12 or C-15 or C-18. The pentadienyl radical formed reacts with a molecule of dioxygen to form a peroxyl radical which undergoes a cyclization and the subsequent radical formed reacts with a second molecular oxygen molecule to generate a hydroperoxide. Finally, a complete reduction yields the whole family of F 4 -NeuroPs with several series (4, 7, 10, 11, 13, 14, 17, and 20) (Fig. 1), when the partial reduction yields the D 4 -and E 4 -NeuroPs (hydroxyketone function of the cyclic ring) (Fig. 2). Note that these metabolites are produced as racemic in vivo.
The increase of the F 4 -NeuroP concentration is demonstrated in several pathologies as for example the neurodegenerative and cardiovascular diseases, and they are now considered as the best markers of the lipid peroxidation. Their quantification in urine and plasma allows a precise, non-invasive and representative measure of oxidative stress (Milne et al., 2007;Mas et al., 2008;Michel et al., 2008;Vigor et al., 2014).
The quantification processes were developed thanks to the availability of synthetic compounds provided by organic chemists able to produce large amount of pure compounds (Jahn et al., 2008). Furthermore, some metabolites that became commercially available were studied and a lot of biological activities were reported over the years (Galano et al., 2017;Ahmed et al., 2020). For example, to determine whether IsoP structures possessed intrinsic biological activities, Morrow et al. (1990) have tested the 15-F 2t -IsoP to study blood pressure. The metabolite was injected in a rat kidney, in the peripheral vein or directly in the kidney, and a reduction of the blood pressure as well as the rate of filtration was observed. IsoPs and other PUFA metabolites thus have biological activities which confer them a role of mediator in a context of OS (Galano et al., 2017;Ahmed et al., 2020).
The lovely story of some of these molecules, especially NeuroPs and dihomo-IsoPs will be presented in this review. Several outcomes in terms of syntheses, diagnosis and biological activities, gathering almost thirty years of research, from organic chemistry knowledge through a smart multi-step synthesis strategy providing many pure compounds, to our fruitful collaborations with a large number of scientists, biologists, clinicians all around the world (Galano et al., 2013(Galano et al., , 2015.

Chemical synthesis
Since the discovery of IsoPs, our group among other developed several strategies to access PUFA metabolites with high purity and enantioselectivity (Vigor et al., 2022).  Nowadays, in our group we are using the last, simple and highly stereocontrolled strategy developed in 2008 (Oger et al., 2008). This strategy is based on a bicyclic a,b-epoxy ketone intermediate (Fig. 4). To this core, and as the key steps, Horner-Wadworth-Emmons and Wittig reactions permit to plug the large diversity of lateral chains, thanks to the synthesis of the appropriate phosphonium salts or phosphonates. The allylic alcohol on the lateral chain is obtained either as (R) or (S)-epimer thanks to an enantioselective chemical reduction. These strategies have allowed us to access IsoPs, dihomo-IsoPs, NeuroPs and other PhytoPs metabolites ( Fig. 4) (Brinkmann et al., 2010;Oger et al., 2010aOger et al., , b, 2012Guy et al., 2014).

New biomarkers
The best technic for the analysis of such metabolites is the tandem chromatography-mass spectrometry, whether using gas or liquid chromatograph (GC-MS, LC-MS). Thanks to the development of MS 2 , GC-MS/MS and LC-MS/MS technics are therefore more specific and sensitive, and allow quantification of particular metabolites found in low abundance in different sample matrices. Both GC-MS/MS and LC-MS/MS have been used for quantification of a wide range of isoprostanoids. However, the derivatization process required for GC-MS analysis to improve the volatility and thermal stability of the compounds is often time-consuming and lead to low recovery yields. Indeed, this derivatization process needs to convert the carboxylic acid into pentafluorobenzyl esters (PFB) by pentafluorobenzyl bromide (PFBBr) in the presence of a catalyst such as N,N'-diisopropylethylamine, and the hydroxyl functions with either O-bis (trimethylsilyl) trifluoroacetamide þ 1% trimethylchlorosilane (BSTFAþTMCS) or N-Methyl-N-trimethylsilyltrifluoroacetamide (Vigor et al., 2014) to form trimethylsilyl ether derivatives. However, derivatization is not a mandatory for LC-MS technic. Therefore, several methods were developed for LC-MS/MS studies of PUFA metabolites (Collado-Gonzales et al., 2015;Lee et al., 2016;Sánchez-Illana et al., 2017;Rund et al., 2018). As for example, our group in collaboration with the group of Justine Bertrand-Michel was able to detect and quantify more than 50 isoprostanoids synthesized in our group, in a run a 20 min (Fig. 5) (Dupuy et al., 2016). It should be mentioned that depending of the series of isoprostanoids, this method permits to separate the two epimers of the allylic alcohol (Fig. 6). In this sense, the development of new technic and/or methods to separate diastereoisomers of PUFA metabolites is still of great interest.

Neuroprostanes
Our two first examples reporting the interest of F 4 -NeuroPs as biomarkers of pathological states were performed by Barden et al. (1996Barden et al. ( , 2004Barden et al. ( , 2012. In a recent study F 4 -NeuroPs, F 2 -IsoPs as well as IsoFs were quantified in maternal plasma and cord blood of women with pre-eclampsia and normal pregnancie. Women with pre-eclampsia had significantly elevated maternal IsoFs and F 4 -NeuroPs, but no F 2 -IsoPs. Cord blood F 4 -NeuroPs were elevated among neonates of mother with pre-eclampsia. Interestingly, cord blood IsoFs were approximately 5-fold higher than those found in maternal plasma. This could reflect the oxidative challenge presented at birth when there is transition from a relatively low intra-uterine oxygen environment to a significantly higher extra uterine oxygen environment. Maternal F 4 -NeuroPs were not significantly correlated with cord blood F 4 -NeuroPs in pre-eclamptic and in normal pregnancies, suggesting the origin of cord F 4 -NeuroPs may be independent of maternal plasma. In normal pregnancy, birth weight was negatively related to maternal F 2 -IsoPs, IsoFs and F 4 -NeuroPs.  T. Durand et al.: OCL 2023, 30, 10 The brain is vulnerable to oxidative insult because of high oxygen requirements for its metabolism and high PUFA composition, in particular DHA. Thus, F 4 -NeuroPs are considered as specific markers of brain OS. Aneurysmal subarachnoid hemorrhage (aSAH) and traumatic brain injury (TBI) are associated with devastating central nervous system (CNS) injury. Two case-controlled studies have shown a significant increase in cerebrospinal fluid (CSF) of IsoFs in aSAH and TBI patients compared with their respective ageand gender-matched controls. aSAH patients also had significantly increased levels of CSF F 4 -NeuroPs and F 2 -IsoPs. Patients with TBI had significantly increased CSF F 4 -NeuroPs but F 2 -IsoPs were similar to control (Corcoran et al., 2011). These data confirm that CNS injury, in case of aSAH or TBI, results in increased OS and as DHA is the brain major PUFA, F 4 -NeuroP levels in CSF could be a much more specific indicator of neurological dysfunction than F 2 -IsoPs. Hsieh et al. (2009) have showed that increased F 4 -NeuroPs in CSF of patients with aSAH correlated with poor neurological outcome. They suggested that F 4 -NeuroPs might be more useful than F 2 -IsoPs in CSF to predict outcome and interpret the role of hemorrhage in aSAH. Although Farias et al. (2008) showed increased F 2 -IsoPs during rat brain ischemia, the E 2 /D 2 -IsoPs were increased to a greater extent, suggesting the latter may better markers of OS in brain ischemia.
The anti-atherogenic effects of omega-3 fatty acids, eicosapentaenoic acid (EPA, C20:5 n-3) and DHA are well recognized but the impact of dietary intake on bioactive lipid mediator profiles remains unclear. Such a profiling effort may offer novel targets for future studies into the mechanism of action of omega-3 fatty acids. Gladine et al. (2014) studied the impact of DHA supplementation on the profiles of PUFA oxygenated metabolites and their contribution to atherosclerosis prevention. A special emphasis was given to the nonenzymatic metabolites knowing the high susceptibility of DHA to free radical-mediated peroxidation, and the increased OS associated with plaque formation. Atherosclerosis prone mice (LDLR 2/2 ) received increasing doses of DHA (0, 0.1, 1 or 2% of energy) during 20 weeks leading to a dose-dependent reduction of atherosclerosis (R 2 = 0.97, p = 0.02), triglyceridemia (R 2 = 0.97, p = 0.01) and cholesterolemia (R 2 = 0.96, p < 0.01). Targeted lipidomic analyses revealed that both the profiles of EPA and DHA and their corresponding oxygenated metabolites were substantially modulated in plasma and liver. Notably, the hepatic level of F 4 -NeuroPs was strongly correlated with the hepatic DHA level. Moreover, unbiased statistical analysis including correlation analyses, hierarchical cluster and projection to latent structure discriminate analysis revealed that the hepatic level of F 4 -NeuroPs was the variable most negatively correlated with the plaque extent (p < 0.001) and along with plasma EPA dihydroxylated metabolites was an important mathematical positive predictor of atherosclerosis prevention. Thus, oxygenated n-3 PUFA, and F 4 -NeuroPs, are potential biomarkers of DHA-associated atherosclerosis prevention. While these may contribute to the anti-atherogenic effects of DHA, further in vitro investigations are needed to confirm such a contention and to decipher the molecular mechanisms of action.

Dihomo-isoprostanes
Rett syndrome (RTT) is a pervasive abnormality of development affecting almost exclusively females, which is included among the autism spectrum disorders. RTT is caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 (MeCP2) genes (De Felice et al., 2012). Although over 200 different MeCp2 mutations have been reported to cause RTT, nine most frequent ones (hotspot mutations) are known to comprise more than three quarters of all the reported pathogenic mutations. The disease shows a wide phenotypical heterogeneity, with at least 4 distinct major clinical presentations, i.e., typical, preserved speech, early seizure variant, and congenital variant. First, clinical evidence indicates that F 2 -IsoPs and F 4 -NeuroPs are involved in the intimate pathogenetic mechanisms of RTT (De Felice et al., 2011). Plasma levels of free F 2 -IsoPs are significantly higher in the early stages of RTT, as compared with the late natural progression of typical RTT. Until 2011, it was thought that the predominant central nervous system damage in RTT occurred in gray matter. However, the relative abundance in myelin of the precursor AdA and the increased level of F 2 -dihomo-IsoPs, strongly confirm an early and severe damage to the brain white matter as suggested by previous brain MRI evidence (De Felice et al., 2011). Thus F 2 -dihomo-IsoPs can be considered early markers of lipid peroxidation in RTT. F 4 -NeuroPs also appear to be important biomarkers in RTT. Plasma F 4 -NeuroPs levels correlate with disease severity in RTT and are significantly related to neurological symptoms severity, mutation type and clinical presentation. Therefore, F 4 -NeuroPs may also play a major role along the biochemical pathway from MeCp2 gene mutation to clinical evidence, proving that a DHA oxidation process occurs (Cortelazzo et al., 2016).

New bioactive lipids
PUFA metabolites are not only biomarkers of lipid peroxidation but also mediators of oxidant injury, and especially the NeuroPs.

Neuroprostanes
It is well reported that an enriched n-3 PUFA diet confers cardioprotective effects due primarily to the two main PUFA, EPA and DHA (GISSI-Prevenzione Investigators, 1999). A large prospective study showed that the most marked effect of DHA and EPA supplementation is a reduction of sudden cardiac death in the months following a cardiac infarction. This benefit has been explained, in part, by a reduction in arrhythmias and systolic cardiac failure. The anti-arrhythmic effects of n-3 PUFA have been confirmed in animal models of cardiac infarction by ligature of the left coronary artery and reported in 2003 by Judé et al. (2003). These and other studies in single cardiac cells have shown that EPA and DHA can modulate the activity of ion channels, the transmembrane proteins responsible for the electrical activity of the heart. However, it has been suggested that oxygenated metabolites of EPA and DHA may also play a role in these actions. In this regard, it has been shown that some of the effect of DHA on rat cardiac ion channels was due to an oxygenated metabolite of DHA, the 4(RS)-4-F 4t -NeuroP (Fig. 7) (Roy et al., 2015). Roy et al. (2015) have tested different F 4 -NeuroPs on arrhythmias induced by isoprenaline and stimulation frequency of isolated ventricular mice cardiac cells. Among them, some F 4 -NeuroPs have anti-arrhythmic effects (IC 50 ≈ 100 nM). The main metabolite, the 4(RS)-4-F 4t -NeuroP, showed potent dose-dependent in cellulo and also in vivo in permanent myocardial infarction (PMI) mice. At the cellular level, the mechanism of action is unlikely to be due to a b-blocker effect, but the anti-arrhythmic property can instead be explained by a rycal-like effect; in particular, stabilization of the RyR2 complex with FKBP12.6 (Andersson and Marks, 2010;Roy et al., 2017).
In 2020, Lee et al. (2020) reported for the first time that native 4(RS)-4-F 4t -NeuroP has a regulatory role in neurons for cell survival on human SH-SYSY neuroblastoma cells. Geng et al. (2022) have also reported a neuroprotective effect of 4(RS)-4-F 4t -NeuroP on primary mouse microglial and BV2 cells and that the 4(RS)-4-F 4t -NeuroP attenuate LPSinduced mitochondrial membrane potential loss in BV2 cells.
Finally, Moretti et al. (2023) reported this year that 4(RS)-4-F 4t -NeuroP possess an effect on human sperm and precisely a role on sperm capacitation mediated again by a ryanodine receptor.
It should be mentioned that to date, there is no biological activity reported for the dihomo-IsoPs.

Conclusion
Our understanding of the role of PUFA peroxidation in the pathogenesis of various human diseases is at an early stage. Regarding DHA, we know that free radical-induced autoxidation of this PUFA occurs in numerous pathological conditions from cardiovascular disorders to cancers and neurodegenerative diseases. Through our knowledge in organic chemistry, we can contribute to clinical and basic research by developing novel synthetic approaches and providing samples for biological and analytical studies. Several new approaches for chiral synthesis of NeuroPs and others isoprostanoids such as dihomo-IsoPs are now available. Some of these products may be used as markers for the diagnosis and management of patients and will need to be measured accurately and precisely. The contribution of each of these unique NeuroPs to tissue and organ damage has to be clearly ascertained within a complex network of signalling molecules and mediators. Despite all the work performed in this field, there is still a lot to discover, and collaborations between chemists and biologists are in this sense highly important.  Page 6 of 8 T. Durand et al.: OCL 2023, 30, 10