## 4 Discussion
### Lipids Levels and Race-Related Trends  
To the best of our knowledge, this is the first report describing half-marathon consequences on circulating sphingolipid metabolism. It is known that exercise may induce modification in plasma levels of numerous circulating biomarkers, inflammatory parameters and lipid metabolites in humans [12] Thus, these changes may affect sphingolipid metabolites as well. In recent years, plasma sphingolipids have attracted attention for their role in pathophysiology of cardiometabolic diseases. In fact, high plasma ceramides may induce endothelial dysfunction, which is closely correlated with aerobic capacity [13], and promote cell growth arrest, cytoskeleton rearrangements, senescence and death (e.g., activation of caspases), impairment of nitric oxide synthase (eNOS) activity and insulin signaling, increasing vascular permeability, oxidative stress and inflammation [14], thus  
Figure 5: Regression of fractalkine and TNF\(\alpha\) in runners (Rho = 0.91 \(p\) = 0.0017 24 h). Green dots—baseline, blue dots—post-race, red dots—24 h post-race. contributing to onset and development of atherosclerosis [15; 16]. Accordingly, sphingolipids may represent an independent risk determinant for ischemic disease [17]. In particular, Cer(d18:1/16:0) appears to be independently correlated with the presence of more vulnerable coronary plaques in CAD patients [18]. Conversely, the Mediterranean diet shows the potential to reduce the adverse effects of high ceramide levels in the PRED-IMED trial (Prevencion con Dieta Mediterraneana, a prospective case-cohort study), which is a study including nearly 1000 elderly subjects at high cardiovascular risk [19]. Ceramide metabolism also appears responsive to the exercise stimulus. Better cardiopulmonary fitness correlated with low ceramide concentration in elderly coronary artery disease patients has been observed [20], whereas muscle ceramide levels decreased after chronic aerobic exercise [21]. Moreover, experimental data suggests that total content of ceramides decreased in the muscle in trained rats, contributing to the elevation of the glucose uptake observed in skeletal muscles after training [22]. Previous data suggested that 12 weeks of aerobic exercise training in obese or diabetic subjects [23] and 16 weeks of exercise in overweight/obese subjects may reduce sphingolipids (e.g., C18:0, C20:0 and C24:1) [24]. A decrease in ceramide concentrations hints at increased insulin sensitivity, which contributes, at least in part, to the beneficial exercise effects [25]. According to these previous results, we also observed a decrease in total ceramides after the race in trained subjects. Thus, all together these data suggest that sphingolipid species can represent valuable mediators of cardiovascular risk. Studies related to cardiac lipotoxicity showed that the lipotoxic species were primarily driven by ceramides and DAG and not triacylglycerol [26]. Regarding DAG species, [PERSON] et al. showed that DAG (32:1, 34:1, 36:2, 36:3) were significantly increased in subjects with high peripheral insulin resistance versus low peripheral insulin resistance subjects [27] In our study, DAG 36:3 decreased significantly after the race, indicating an improvement of lipotoxicity, attributable to that species after the acute exercise. Experimental data suggest that total content of ceramides decreases in the muscle of trained rats, contributing to the elevation of the glucose uptake observed in skeletal muscles after training [22]. In overweight, obese subjects or type 2 diabetes (T2D), there is a decrease in total ceramides, indicating that endurance training reduces total content of ceramides, likely improving glucose tolerance [22; 25; 27; 28; 29]. However, ceramide metabolism in patients and obese subjects could be dissimilar to that in lean, trained individuals. In fact, the increased lipid metabolism following the physical exercise reduces the substrate availability required for ceramide synthesis (e.g., palmitate, myristate).