*3.3. Oxylipins*

Like other oxidized lipids, traditional analytical methods have been widely used for assessing oxylipins [55]. Miler et al. (1985) developed enzyme-linked immunosorbent assays (ELISA) to assess LTC4, LTB4, 6-keto PGF1 alpha, and TXB2 [56]. The main drawback of this approach is that only one analyte can be targeted with one set of analysis. GC/MS has also been utilized to measure oxylipins, for instance, Tsukamoto et al. (2002) developed a method to measure oxylipins including PG, isoprostane and TXs with GC/MS [57]. Due to the complex sample preparation and thermal decomposition during derivatization, HPLC based methods have been used recently for oxylipin analysis [55].

HPLC/ESI-MS has been utilized to quantify plasma oxylipins in patients [18,58]. For instance, Caligiuri et al. (2017) quantified 39 plasma oxylipins in patients with PAD using HPLC/ESI-MS [18]. Among all the identified/quantified oxylipins, 4 oxylipins were significantly correlated with the presence of cardiovascular/cerebrovascular events. For instance, plasma levels of 8,9 DiHETrE were significantly elevated in patients with ACS when compared with ones without ACS (0.3 ± 0.1 versus 0.2 ± 0.0 nM, respectively). Plasma concentrations of PGE2 were significantly higher in patients with angina when compared with subjects without angina (0.4 ± 0.0 versus 0.3 ± 0.0 nM, respectively). Moreover, they found that 16-HETE, TRX B2, and 11,12- DiHETrE increased the odds of having cardiovascular/cerebrovascular events in this population.

To prepare samples for lipidomic analysis, oxylipins can be extracted using liquid–liquid extraction and/or solid-phase extraction procedures. Use of chloroform/methanol mixtures, according to Bligh and Dyer, is the most common liquid–liquid extraction protocol for oxylipins. In this method, oxylipins are dissolved in organic solvents, but hydrophilic materials such as proteins are eliminated following phase separation. Solid-phase extraction can be conducted using commercial columns pre-packed with various sorbents. Reverse-phase HPLC with a C18 column has been used widely to separate oxylipins [18]; however, Zu 2016 et al. used ultra-performance liquid chromatography (UPLC) (C18 column) to separate oxylipins before analysis with MS [15]. The UPLC column has better resolution, lower detection limits, and a shorter chromatographic run when compared with HPLC [51]. Deuterated oxylipins are commercially available, which are used as internal standards. These standards are matched with groups of endogenous oxylipin species in terms of chemistry, retention time, and ionization efficiencies [51]; as mentioned previously, internal standards are needed to assess the extraction efficacy and instrument response [51].

Tandem mass spectrometry is the most sensitive system for analyzing oxidized lipids, particularly when predetermined species are desired, and is known as the targeted approach. Multiple-reaction monitoring (MRM) is an acquisition mode that monitors the transition of a selected precursor ion, based on the mass/charge value, to a specific product ion using the fragmentation pattern. It has been reported that by using separation techniques such as UPLC and MRM transitions, more than hundreds of oxylipins can be identified/quantified in a single acquisition at picogram/fentomole levels [59].

Quantifications of oxidized lipids can be carried out by generating calibration curves for internal standards. As mentioned previously, deuterated oxylipins are commercially available, which can be used as oxylipin internal standards. However, non-oxidized PLs and lyso PL (LPL) such as PI (31:1), PC (9:0), and LPC (17:0) have been used as internal standards for OxPI and OxPC quantifications as there is no deuterated standard for OxPL analysis.

#### **4. Role of Bioactive Lipids in ACS**

#### *4.1. 4-HNE and MDA*

Previous studies have shown that 4-HNE may contribute to many cardiovascular diseases (CVD) [60–62]. It can be generated during Ox-LDL oxidation and makes apo B-adducts, which are identified by scavenger receptors, leading to elevated uptakes of Ox-LDL by macrophages and the formation of foam cells. Previous studies were able to identify HNE-adducts in human atherosclerosis lesions by using anti-HNE antibodies [63,64]. The role of HNE in ACS has not been studied well; however, a study by Gargiulo et al. (2015) showed that HNE may induce plaque instability by increasing the expression and synthesis of inflammatory cytokines such as interlukine-8 (IL-8), interlukine1-β (IL-1β), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-9 (MMP-9) via Toll-like Receptors 4/Nuclear Factor-κB (TLR4/NF-κB) signaling pathways [65]. In addition, a recent study showed that levels of HNE in coronary sinus were significantly higher in STEMI patients before and after percutaneous coronary intervention (PCI) when compared with patients with stable ischemic heart disease (IHD) who underwent elective PCI [33].

In the last 30 years, numerous studies have extensively shown that elevated levels of MDA are associated with CVD. Having traditional CVD risk factors such as cigarette smoking [21,66], hypertension [66], hyperlipidemia [67,68], and diabetes [69,70] were reported to be significantly correlated with higher MDA levels. Increased levels of MDA have been reported in plasma of patients with atherosclerotic diseases [71]. A nested case-control cohort showed that LDL-MDA was a strong predictor of carotid wall thickness in hypercholesterolemic men [72]. In a perspective study with 634 patients having CVD, serum levels of MDA were strong predictors of cardiovascular events (including MI, stroke, hospitalizations for non-fatal cardiovascular events mainly UA), and major vascular procedures (percutaneous transluminal coronary angioplasty (PTCA)/coronary artery bypass grafting (CABG)), independent of traditional risk factors such as blood pressure (BP), total cholesterol, high-density lipoprotein-cholesterol (HDL-cholesterol), LDL-cholesterol, triglycerides (TG), age, gender, body mass index (BMI), and inflammatory markers in patients with coronary heart disease (CHD) [73]. In a study by Bagatini et al. (2011), increased levels of MDA were observed in MI patients and subjects with CVD risk factors (including cigarette smoking, hypertension, and family history of CHD) when compared to healthy controls [74].

#### *4.2. OxPL*

Atherogenicity of OxPLs was shown by Hörkkö et al. (1999) as they found that OxPLs contribute to Ox-LDL recognition by macrophages. They also found that the monoclonal antibody E06, which binds to the phosphocholine head group of PLs on Ox-LDL, inhibits Ox-LDL uptakes by macrophages [75]. Since then, several studies have shown that OxPLs may have roles in various steps of atherosclerosis such as facilitating Ox-LDL uptake by macrophages [76], mediating cellular inflammatory responses [77], and stimulating angiogenesis [25].

1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), and 1-palmitoyl-2-glutaroylsn-glycero-3-phosphorylcholine (PGPC), which are derived from arachidonyl phosphatidylecholines, are produced during Ox-LDL modification and have been identified in atherosclerotic plaques [78,79]. These fragmented OxPLs are toxic and create tissue injury through inflammatory responses [77] and apoptosis [80]. LP (a) is the main carrier of OxPLs in plasma, although they can also be transferred by LDL and HDL [81]. Previous studies have demonstrated that levels of OxPLs are strongly correlated with LP (a) levels and the extent of coronary stenosis [82,83]. Therefore, it has been suggested that the atherogenicity of LP (a) can be attributed to OxPLs as its a carrier of proinflammatory oxidized molecules.

Tsimikas et al. (2003) developed a method to measure OxPL by using murine monoclonal antibody E06 [13]. This antibody binds to the phosphorylcholine (PC) head group of OxPL, particularly POVPC. Therefore, the amount of PC-OxPL per apoB-100 (OxPL/ApoB) containing lipoproteins can

be calculated. By using this approach, they showed that OxPL levels increased significantly in MI patients after PCI, suggesting that these compounds are released and/or generated as a result of plaque rupture [13,84]. Prospective studies have shown that OxPLs levels can be considered as biomarkers of atherosclerosis progression, cardiovascular death, MI, and stroke. In a prospective Bruneck study, the 5-year follow-up of 700 participants aged 40 to 79 years old showed that OxPLs levels were strongly and significantly associated with the presence, extent, and development of carotid and femoral atherosclerosis, and predicted the presence of symptomatic CVD [85]. The ten-year follow-up of this population showed that risk of cardiovascular events, which was defined as cardiovascular death, MI, stroke, and transient ischemic attack (TIA), were significantly elevated in participants in the highest tertile of OxPLs/apoB than those in the lowest tertile independent of traditional risk factors, suggesting that OxPLs/apoB levels may predict the risk of 10-year CVD events [86].

#### *4.3. Oxylipins*

Pioneering studies have shown the association between oxylipins derived from AA with UA and atherosclerosis. Elevated levels of TXB2 have been reported in the coronary circulation of patients with unstable angina [87,88]. Moreover, Mallat Z et al. (1999) showed that HETEs levels were significantly higher in plaques obtained from symptomatic patients (with unstable plaque) versus patients with stable plaques [89]. Similarly, Waddington et al. (2001) found higher levels of 15-HETE and 11-HETE in atherosclerotic plaque retrieved from individuals undergoing carotid endarterectomy [90]. Recently, a targeted metabolomics study showed that among all identified metabolites, 20-HETE was the only compound that was significantly higher in patients with atheroma plaque when compared with healthy subjects [91].

New studies have also investigated the role of oxylipins in the diagnosis and prognosis of ACS and MI. A retrospective nested case-control study, comprised of 470 ACS patients and 39 subjects without CHD as a control group, was conducted in a Chinese population [15]. Among the ACS patients, subjects who had had a major adverse cardiovascular event (MACE) during the 1037 days of follow up period were identified as the MACE group, and ACS patients without MACE during this period were named as the non-MACE group. In this study, LTB4, 8-HETE, 11-HETE, 12-HETE, and 15-HETE were significantly elevated in the ACS patients (both the MACE and non- MACE groups) when compared with the controls. In addition, the levels of 5-HETE and 9-HETE were significantly higher in the MACE group when compared with the controls, suggesting the potential diagnostic value of these oxylipins in ACS. In addition, the levels of 20-HETE were significantly elevated in the STEMI group when compared with the non-STEMI group, indicating that the pathogenesis of STEMI and non-STEMI may be different. Moreover, the 19-HETE levels, a vasodilator oxylipin, were significantly lower in the MACE group than the non-MACE and control groups. ACS patients who had higher levels of 19-HETE (higher than 0.13 ng/mL) tended to have better prognosis (up to 72%) than those with lower levels [15]. In a prospective study by Sun et al. (2016) [92], the association between oxylipins and the incidence of MI was investigated in 744 AMI cases and 744 matched controls, aged 47–83 years within the Singapore Chinese Health Study. They found inverse correlations between pro-thrombotic TXB2 and AMI risk, and suggested that this unexpected association was more related to sample collection, processing, and storage conditions than biological differences. Moreover, in this study, only 19 oxylipins, which had potential roles in inflammation, blood pressure, and platelet degranulation were measured, and not the full spectrum. In a study by Caligiuri et al. (2017), the associations between oxylipins and the occurrence of cardiovascular/cerebrovascular events, defined as STEMI, non-STEMI, and UA, in 24 patients with peripheral artery disease (PAD) were assessed. They found that levels of 16-HETE, TXB2, and 11,12-DiHETrE were significantly associated with increased odds of cardiovascular/cerebrovascular events in PAD patients and showed that with every 1 nmol/L increase in 8,9-DiHETrE concentrations, the odds of ACS increased by 454-fold. In this particular study, 8,9-DiHETrE elevated the odds of ACS by 92-fold [18].

All of the clinical studies that have assessed these bioactive lipids in ACS patients are presented in Table 1.



### **5. Conclusions**

There is accumulating evidence that bioactive lipids play roles in ischemic cardiovascular disease. We have made great strides in elucidating their activity by utilizing antibody based approaches. Given the advances in mass spectrometry, we were able to identify and quantitate individual oxidized lipids in plasma. It is important that we standardize the current mass spectrometric methods of quantitation and analysis, so that large cohorts of patients can be analyzed. This would lead to a better understanding of the specific contribution of each lipid molecule to the overall pathophysiology.

**Author Contributions:** Writing—Original Draft Preparation, Z.S.; Writing—Review & Editing, A.R. **Conflicts of Interest:** The authors declare no conflicts of interest.
