2.1 Classic lipid parameters
Prior to the lipidomic study, a classic lipid profile was obtained. Fig. 1 shows the box plots of measurements of total cholesterol (TC), high-density lipoprotein - cholesterol (HDL-C), low-density lipoprotein – cholesterol (LDL-C) and triglycerides (Tg) in each group of samples. As expected mainly changes were observed for TC and LDL-C.
2.2 Lipids and FFAs evaluation
Fig. 2 shows the plasma concentrations of the analyzed lipids, categorized by classes (CE, CER, FFA, LPC, LPE, PC, PE, SM, and TAG) (Fig. 2a) and by groups (G1, G2, G3 and G4) (Fig. 2b). Comparing the mean values in each group, it is possible to observe that all metabolites were decreased in both groups after the exposure to the treatments at D30, except for LPC, LPE and TAG.
Considering rosuvastatin administration at D30, there was a decrease for CE (40%), CER (40%) and SM (36%), PC (31%), PE (26%), and FFA (20%). However, LPC was the only lipid increased (23%) in G2. Following simvastatin plus ezetimibe therapy at D30, there was a decrease in PE (36%), SM (25%), PC (23%), CE (21%), CER (10%) and FFA (11%). LPC, TAG and LPE were almost inaltered in G4. Comparing the two arms of treatment at D1 (G1 and G3), it is possible to observe similar lipid and FFA level reduction in G3, except for CE and CER; CER was 29% less abundant in G3, followed by CE (20% less), SM (14% less), LPE (11% less), FFA (10% less), and PC (7% less). Regarding the effects of treatments at D30 (G2 and G4), LPC and PE were 13% less abundant in the simvastatin plus ezetimibe arm (G4), while TAG was 32% more abundant.
2.3 Correlation studies
Levels of LDL-C, HDL-C, TC, and Tg were correlated with levels of lipid classes (CE, CER, FFA, LPC, LPE, PC, PE, SM, and TAG) for all studied patient groups (Fig. 3). Fig. 3a presents the correlations between the two therapies at D1 of STEMI. Opposite correlations between the clinical parameters and some lipids (PE, TAG, LPE, and CE) were observed.
Considering rosuvastatin administration, PE was negatively correlated with CT, HDL-C and LDL-C and positively correlated with Tg; TAG was negatively correlated with HDL-C, but positively correlated with Tg; LPE was negatively correlated with LDL-C and positively correlated with HDL-C; and CE was positively correlated with Tg. Fig. 3b depicts correlations between the two therapies at D30. Opposite correlations between the clinical parameters and some lipid classes were also observed. However, besides PE, TAG, LPE, and CE, PC, SM, and CER also presented contrasting correlations with the clinical parameters at D30.
Considering simvastatin plus ezetimibe administration, PE was negatively correlated with CT and HDL-C; TAG was negatively correlated with TC and HDL-C, but positively correlated with Tg; LPE was negatively correlated with HDL-C; CE was positively correlated with TC and LDL-C and Tg, but positively correlated with Tg; PC was negatively correlated with CT and HDL-C; SM was negatively correlated with TC, HDL-C and LDL-C; and CER was positively correlated with Tg.
Figure 3 shows different behaviors among some clinical parameters with lipids at D30. For instance, alterations in LDL-C and Tg levels related to PE were not observed at D30 in both lipid-lowering therapies and the same was observed for LDL-C levels regarding LPE.
With rosuvastatin, HDL-C levels related to LPE, which were positively correlated at D1, became negatively correlated at D30. In addition, TC and HDL-C levels related to PC and SM became negatively correlated with CE, as well as LDL-C levels related to SM, whereas, Tg levels related to CT, LDL-C, and LPEbecame positively correlated. With simvastatin plus ezetimibe at D30, Tg levels related to TC, LDL-C, LPE, PC and SM became positively correlated and with rosuvastatin at D30.
2.4 Identification of statistically significant metabolites
2.4.1 Univariate and multivariate analyses
Data quality was carried out by inspecting the repeatability of lipids and FFAs in the QC plasma sample, analysed throughout data acquisition. More than 80% of the quantified metabolites in the QCs have acceptable coefficient of variation porcentages (% CV) for peak areas; in this work, < 20% CV was used as a criterion to retain that particular component in the dataset for further evaluation, which were in agreement to the recomendation12, 13.
The OPLS-DA scores plot (Fig. 4) shows that patients of G1, G2, G3 and G4 are clustered and they have distinct lipid profiles from each other (R2 = 0.231; Q2 = 0,246; p = 0,032). The discriminant variables were obtained by multivariate (VIP > 1) and/or univariate evaluation (p-value and q-value < 0.05) with 95% confidence interval. These findings with their respective increasing or decreasing percentage related to each studied group are better visualized in Fig 5 and S2. Patients of G1 are clustered due LPC, CE, CER, FFA, PC, PE, and SM alteration levels. LPC(20:4/0:0) presented the largest contribution, with 80% and 74% decrease related to G2 and G4, followed by PE(16:0/18:2), with 57% and 55% increase related to G2 and G4, respectively; SM (d18:1/20:0) with 50% increase related to G2, and 29% and 55% related to G3 and G4, respectively; PC(16:0/18:2) with 48% increase related to G2 and 46% related to G4; PE(18:0/18:2) with 47% increase related to G2 and 45% related to G4; CE(18:2) with 45% increase related to G2 and 33% related to G4 and PC(18:1/20:4) with 41% and 45% decreased related to G2 and G4. The others, PC, PE, CER, SM and FFA presented a range from 7% up to 44% increasing. However, patients of G2 are clustered manly, because,LPC was the metabolite that presented the highest increased levels, being LPC (20:4/0:0) 80% increased related to G1 and 32% related to G3; LPC (17:0/0:0) 56% increased related to G1, 34% related to G3 and 20% related to G4; LPC (16:1/0:0) 52% increased related to G1 and 43% related to G3, and LPC (18:1/0:0) was increased 50% related to G1 and 33% related to G4. Lauric acid (12:0) level slightly increased in G2: 18% related to G1, 22% and 14% related to G3 and G4, respectively, and PC(18:1/20;4) was increased 41% related to G1 and 13% related to G3.
In addition, Figure 4 shows that patients of G3 are clustered due the increased levels in TAG, CE, SM, PC and FFA, being the higher increasing in TAG50:2-FA18:2 and CE (22:5), with increase of 113% and 65% related to G2, respectively. SM (d18:1/16:0) and oleic acid (18:1) presented almost the same contribution, with an increasing of 50% and 47% related to G2, and 34% and 30% related to G4, respectively. The other FFAs and PCs presented an increase range from 2% up to 34%. However, the major increased levels in G4 were observed in TAG and LPC, being the major and similar increased levels in TAG56:5-FA20:4, TAG56:4-FA20:3 and LPC(20:4/0:0), with increase of amost 75%, followed by TAG56:5-FA20:3 and TAG56:5-FA18:0 with an increase of 68% and 64% related to G1, respectively. The other TAGs and PC presented an increase range from 1% up to 57%. SM (d18:1/16:0) decreased, with 30%, 1% and 34%, respectively, related to G1, G2 and G3. Fig. 6 presents the discriminant metabolites and their similarity for each group. LPC(20:4/0:0) and PC(18:1/20:4) levels were similar in G1, G2 and G4. However, SM (d18:1/16:0) levels was similar to G1, G3 and G4.