Accurate measurement of RCs is difficult due to the heterogeneity of lipoprotein composition, size and density, as well as to their rapid metabolization. RC measurement by separating lipoproteins with ultracentrifugation or polycrylamide gel electrophoresis is complex and is rarely performed in the clinical setting. Conceptually, RCs represent the non-HDL and non-LDL circulating cholesterol. In both clinical practice and epidemiology, and only in fasting conditions, it has been proposed to roughly calculate plasma RCs levels by subtracting HDL-C and LDL-C values from total cholesterol. Excluding severe hypertriglyceridemia, where Frieldwald formula [26] is inapplicable, RCs values are easily computable starting from routine laboratory values [26]. In absence of dosed LDL-C, considering the 1 to 5 ratio between cholesterol and triglyceride content in VLDL under fasting conditions, we can easily calculate RCs dividing serum triglycerides by 5. Recently, a new method for calculating fasting LDL-C has been proposed, which allows a more accurate estimate of the values than those derived from the Friedwald formula [26]. This method is useful in conditions of serum low LDL-C and high TG which are observed with increasing frequency because of the increasingly effectiveness of LDL-lowering therapies and the growing prevalence of obesity and metabolic syndrome (19). This new calculation method suggests applying a correction factor to VLDL C/TG ratio, which in the Friedwald formula is calculated as 1:5, based on the individual values of serum non-HDL C and TG [27].

In addition to calculation methods, RC can be directly measured using a variety of analytical methods including ultracentrifugation, nuclear magnetic resonance (NMR) spectroscopy, and by a direct automated assay. A quantitative method for assessing the concentration of RC in fasting conditions is based on the immunoseparation of remnant-like particles in serum using monoclonal antibodies to apo B-100 and apoA1 [28]. The measurement of RC derived from the exogenous pathway (i.e., chylomicron remnants) can be performed via ultracentrifugation or, indirectly, by measuring apoB-48. Recently, within the Copenhagen General Population Study, it has been demonstrated that directly measured RC identifies more accurately patients at higher risk of myocardial infarction, in comparison to calculated ones [29].

In non-fasting conditions, RC evaluation is more complex since remnants deriving from intestinal chylomicrons are added to VLDL remnants. In this condition, RCs are strongly correlated with TG serum levels which vary according to lipid meals composition and to fasting length since the last meal. In fact, after a fatty meal, apo-B48 levels, TGRLs-C and TG rapidly increase, while there is no postprandial increase in the apoB-100 and LDL-C values. For this reason, postprandial apoB-48 values could be used as a very sensitive marker of non-fasting triglyceridemia, using immunoturbidimetric and immunonephelometric assays commercially available on commonly used automated systems from several manufacturers [30].

RC serum levels vary little between fasting and postprandial periods. Therefore, even in non-fasting condition, RC levels can be accurately calculated by subtracting HDL-C and LDL-C from total cholesterol. To date, RCs in most studies have only been evaluated in fasting conditions and we have little information regarding RC and LRT concentration during the postprandial period. Both increase after a fatty meal, mostly in subjects with primary or secondary lipoprotein metabolism alterations and in those with insulin resistance. However, at the present, we do not yet have a standardized oral fatty meal test for the evaluation of lipid changes during the postprandial phase.

Finally, there is a strong urgency for the development of automated direct analysis methods given the importance of remnants evaluation in the definition of residual cardiovascular risk [30].

Remnant lipoproteins are cleared by the liver mainly via LDL receptor or penetrate arterial wall, thus contributing to the initiation and progression of atherosclerotic lesions. Unlike chylomicrons and VLDLs which do not penetrate the vessel wall due to their large size, and similarly to LDLs, LRTs remnants, due to the small size, easily pass through the arterial wall and are retained in the sub-endothelium, where they bind to the connective matrix and promote inflammation. Their presence stimulates the progression of smooth muscle cells and the proliferation of resident macrophages [31,32]. However, unlike LDLs, TGRLs are directly degraded by scavenger receptors in macrophages in the absence of oxidative modifications thus contributing to plaque formation and progression. RCs can also induce endothelial dysfunction, the production of adhesion molecules and platelet activation, as well as increase the expression of CD40 and metalloproteins [33,34]. Finally, high levels of non-fasting RCs can increase the expression of inflammatory interleukins and cytokines, increase the inflammatory response of monocytes and induce the appearance of a chronic low-grade inflammation, which, on the contrary, is not observed in the presence of high levels of LDL-C [6,35].

Several studies examined the atherogenic role of RCs. Since 2001, a positive association was described, independent of traditional risk factors, between TGRLs-remnant levels and the ischemic heart disease prevalence in a sample of women from the Framingham Heart Study [36]. Subsequently, Fukuscima et al. [37] demonstrated that elevated TGRLs-C levels were a significant and independent risk factor for ischemic heart disease and type 2 diabetes mellitus.

However, it is in Denmark that the largest studies on the predictive role of TGRLs on cardiovascular events have been carried out. In a study conducted over 22 years on 90,000 Danish subjects, the progressive increases in non-fasting measured RC and LDL-C were similarly associated with the increased risk of ischemic heart disease and myocardial infarction; however, only RC levels were associated with all-cause mortality [50,51]. It was estimated that a 1 mmol/L (39 mg/dL) increase in RC in non-fasting conditions was shown to correspond to a 2.8-fold increased risk for coronary heart disease [50,51]. A subsequent study, also conducted in Denmark on a court of 5414 patients with ischemic heart disease, confirmed the association between calculated and/or measured non-fasting RC levels and all-cause mortality, while a similar association was not observed for LDL-C levels [4]. More recently, lowering RCs of 32 mg/dL and 81 mg/dL was estimated to reduce recurrent MACE by 20% and 50% respectively [52]. Finally, very recently, in a follow up study of 19,650 adults in the United States from the National Health and Nutrition Examination Survey (NHANES) (1999–2014), elevated RC levels were independently associated with cardiovascular mortality [53].

Similarly, in a series of patients with coronary heart disease treated with lipid-lowering therapies and reaching LDL-C values < 100 mg/dL, TGRL-C values predicted coronary events recurrence, suggesting RCs as a possible new target of “residual risk” [38]. Similar results were reported by Kugiyana C. et al. in 153 patients with coronary heart disease [40]. Moreover, in a TNT trial post-hoc analysis, fasting TGRLs-C values predicted cardiovascular events and 1 SD RC reduction by atorvastatin reduced cardiovascular events incidence similarly to that obtained with 1 SD LDL-C reduction [54].

The association between RCs and cardiovascular disease was confirmed in different ethnicities). In a study conducted in Japan, confirming the predictive role of CR levels on cardiovascular events recurrence in secondary prevention, RC values were used to increase the prognostic value of traditional risk factors, allowing the authors to better stratify patients’ cardiovascular risk [41]. In addition, no differences were observed in the predictive role between USA black (Jackson Heart Study) and white (Framingham Offspring Cohort Study) subjects [42]. In a Chinese community-based population, increased RC levels better associated with new-onset carotid plaque diagnosis in comparison to other conventional lipid parameters [47]. Finally, in ASCVD-free individuals from three landmark US cohorts—the Atherosclerosis Risk in Communities (ARIC) study, the Multi-Ethnic Study of Atherosclerosis (MESA), and the Coronary Artery Risk Development in Young Adults (CARDIA)—levels of RC were associated with ASCVD independently from traditional cardiovascular risk factors, such as LDL-C, and non-HDL-C or apo-B levels [45].

The good RCs performance for the stratification of cardiovascular risk in dysmetabolic patients was confirmed by several studies. The predictive role of RCs on CV events was confirmed in subjects with type 2 diabetes and chronic kidney disease [43]. Moreover, in a prospective observational cohort study including 798 unselected NAFLD patients with cardio-metabolic diseases, high RC levels were predictive of future CVD [46]. Similarly, in the PREDIMED study, in overweight or obese subjects at high cardiovascular risk, TG and RC serum levels, but not LDL-C, were associated with cardiovascular outcomes [44]. Recently, a retrospective registry of real-world patients treated with PCSK9 inhibitors demonstrated a positive effect on RCs and lipid residual risk beyond LDL-C reductions [55].

Recently, data from Copenhagen General Population Study suggested a stronger association of elevated RCs with peripheral artery disease, than that observed with coronary heart disease and ischemic stroke [48]. Data from the same population also showed that high RCs and triglyceridemia associate with cardiovascular and other non-neoplastic causes of death [49].

Interestingly, mixed results were reported using lowering triglycerides (and cholesterol remnants) drugs to reduce cardiovascular events. While the use of pemafibrate was not associated with cardiovascular benefits [56], icosapent ethyl, was found to be effective in reducing both triglycerides and cardiovascular disease incidence [57]. In addition, other large trials, which used different formulations of omega-3 fatty acids, did not report significant cardiovascular risk reduction [58]. Some authors hypotizes a putative anti-inflammatory effect of icosapent ethyl. While the ANCHOR study showed a reduction of inflammatory markers in patients with decompensated diabetes [59], data from the REDUCE-IT did not confirm this finding [60]. A wide debate on the causes of these contrasting findings is still ongoing [61].