Extensive evidence has consistently substantiated the pivotal role of low-density lipoprotein (LDL) in both the initiation and advancement of atherosclerotic cardiovascular disease (ASCVD) (1–4). Consequently, the measurement of LDL levels is paramount in the comprehensive evaluation of cardiovascular risk, as emphasized in universally recognized international guidelines (5, 6).

Lowering LDL-C remains the cornerstone of ASCVD prevention and treatment. However, clinical observations reveal a paradox: some individuals with moderately elevated LDL-C remain free of ASCVD, while others with low LDL-C still experience major cardiovascular events such as ST-elevation myocardial infarction (STEMI) and stroke (7, 8). These discrepancies highlight the need to look beyond total LDL-C levels to identify the truly atherogenic subfractions or variants of LDL, including small, dense LDL (sdLDL), oxidized LDL (oxLDL), lipoprotein(a) [Lp(a)], and electronegative LDL [L5/LDL(-)] (9). While LDL-C levels and subfraction characterization provide valuable insights into atherogenic processes, they do not fully account for the residual cardiovascular risk that persists despite optimal lipid-lowering therapy (10). Additional factors such as inflammation, elevated Lp(a), triglyceride (TG)-rich lipoproteins, and non-lipid contributors play important roles in ASCVD pathogenesis, underscoring that the study of LDL subfraction should be integrated into a broader, multifactorial risk assessment strategy (11).

Several research studies have provided evidence demonstrating a correlation between distinct subgroups of LDL and the occurrence of atherosclerosis (AS). Specifically, sdLDL has been identified as not only closely related to AS but also exerting a more pronounced impact (12). oxLDL is toxic toward endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts; proliferating cells are more susceptible than quiescent cells in the S-phase of the cell cycle, i.e., during DNA synthesis (13). Lp(a) has been thought to encompass a spectrum of potential deleterious effects, including pro-atherosclerotic, prothrombotic, and pro-inflammatory roles (14). L5/LDL(-), on the other hand, has been established as a dynamically active participant in the atherosclerotic process and is considered a potential inflammatory biomarker (15). The purpose of this review is to investigate the interrelationship between sdLDL, oxLDL, Lp(a), and L5/LDL(-) levels and their association with AS.

LDL is generally characterized as a lipoprotein fraction with a density ranging from 1.019 to 1.063 g/ml, which can be separated using different laboratory techniques (16). The LDL fraction is not uniform and can be divided based on physicochemical properties such as density, size, and composition, with specific subfractions like sdLDL, oxLDL, Lp(a), and L5/LDL(-) linked to an increased risk of ASCVD.

There are several methodologies used for assessing LDL particle density, size, and electronegativity (Table 1).

Density gradient ultracentrifugation technique separates LDL particles into subfractions based on their density, typically divided into three to four subclasses. Another ultracentrifugation method, iodixanol gradient ultracentrifugation, is a variation that uses iodixanol instead of salt gradients, providing slightly different density ranges for LDL subfractions (52).

Gradient gel electrophoresis separates LDL particles based on size under non-denaturing conditions. LDL subclasses are typically defined as: LDL I (large): 26.4–28.5 nm; LDL II (intermediate): 25.5–26.4 nm; LDL III A and B (small): 24.2–25.5 nm; LDL IV A and B (very small): 22.0–24.2 nm (19). Two phenotypes are distinguished based on peak LDL particle diameters: Pattern A: >25.5 nm (large and intermediate LDL), Pattern B: ≤25.5 nm (small and very small LDL).

Nuclear magnetic resonance (NMR) spectroscopy is a laboratory technique used to directly measure LDL particle number and size distribution (53). By providing detailed information on lipoprotein particle size, concentration, and composition, NMR-derived lipoprotein profiles have become a valuable adjunct to cardiovascular risk assessment. Precipitation methods separate sdLDL from larger LDL particles using different reagents (54, 55). Specifically, heparin-magnesium precipitation separates sdLDL using detergent and sphingomyelinase treatment (56). Dynamic light scattering measures LDL particle size and can be used to assess LDL aggregation susceptibility (56). Anion exchange chromatography is used to separate LDL according to its electronegativity which separates L5/LDL(-) from native LDL (57). As a high-resolution electrophoretic technique, capillary isotachophoresis can also be used to assess LDL electronegativity (58).

Some studies combine these techniques to comprehensively evaluate LDL properties, including particle density, size, and electronegativity (59). These approaches often integrate ApoB measurements to estimate LDL particle number alongside TG levels for assessing particle size. While direct LDL-C quantification methods exist, they are less commonly used due to higher costs, leading many studies to rely on indirect estimations like the Friedewald equation (60). Importantly, these analytical methods focus on LDL characterization for their atherogenic potential and should not be confused with the clinical concept of the LDL Window, which refers to the duration of LDL reduction after apheresis therapy.

The size variability and density distribution of LDL particles plays a crucial role in determining their atherogenic potential, and understanding this heterogeneity is key to elucidating the complex mechanisms underlying CVD. As mentioned, LDL particles are not uniform; they exhibit a broad range of sizes and densities that influence their biological behavior, metabolism, and interaction with the arterial wall. This variability has significant clinical implications, as different LDL subfractions contribute to AS in distinct ways.

sdLDL particles are often associated with a greater atherogenic risk compared to larger, buoyant LDL particles due to several mechanistic properties, such as a greater propensity for arterial wall retention and increased susceptibility to oxidation (61). Increased proportion of sdLDL is associated with higher cardiovascular risk, even when total LDL-C levels are within normal range (62). The presence of sdLDL is often part of an atherogenic lipoprotein phenotype, which includes elevated TGs and low HDL cholesterol (HDL-C) (63). Studies have demonstrated that sdLDL is an independent risk factor for CVD, emphasizing the importance of both LDL quantity and quality in assessing cardiovascular risk (64). It is important to note that while this density-based classification is widely used, some recent studies have challenged the notion that all large LDL particles are less atherogenic. For instance, one study found that both very small and very large LDL particle sizes were associated with increased mortality risk compared to intermediate-sized LDL (63). sdLDL particles are also distinguished by their longer plasma residence time due to a reduced affinity for LDLR, which decreases their clearance from the circulation. This prolonged exposure time allows for increased interaction with arterial proteoglycans, promoting their retention and subsequent uptake by macrophages (1). Furthermore, these particles exhibit altered lipid and protein composition, such as a higher ratio of TG to CEs, which may render them more prone to oxidation, desialylation, and glycation modifications, thereby enhancing their atherogenic potential (65).

One of the primary characteristics that enhances the atherogenicity of sdLDL particles is their increased ability to penetrate the endothelial barrier and accumulate in the subendothelial space (66). Once trapped, these particles are more likely to undergo oxidative modification, forming oxLDL, which is a potent pro-inflammatory agent and a key contributor to plaque formation and instability. oxLDL is recognized by scavenger receptors on macrophages, leading to the formation of foam cells and the initiation of an inflammatory cascade within the arterial wall (67). This sequence of events is a critical step in the development and progression of atherosclerotic plaques, highlighting the importance of LDL particle size in influencing disease outcomes.

The relationship between LDL particle size and cardiovascular risk has been substantiated by several epidemiological and clinical studies. Individuals with a predominance of sdLDL particles are more likely to exhibit atherogenic dyslipidemia, characterized by elevated TGs, low HDL-C, and an increased number of LDL particles. This pattern, often observed in patients with metabolic syndrome and type 2 DM (T2DM), is associated with a higher risk of CAD and other cardiovascular events (CVEs) (68). Recent meta-analyses confirm sdLDL particles are independently associated with an increased risk of CVEs, even after adjusting for traditional risk factors such as total LDL-C levels (64). As mentioned earlier, advanced lipid testing methods, such as NMR spectroscopy and ion mobility analysis, have enabled more precise quantification and characterization of LDL subfractions. These technologies allow for the identification of sdLDL particles and provide valuable information on their concentration and distribution within the LDL particle spectrum. Incorporating these measurements into routine clinical practice could improve risk stratification and enable more targeted therapeutic strategies for managing dyslipidemia and preventing CVD (69). However, due to the substantial discrepancies between NMR-derived sdLDL concentrations and those obtained via traditional methods such as ultracentrifugation or GGE, with NMR generally yielding higher values, it is necessary to carefully calibrate or validate NMR methodologies to ensure comparability across platforms.

Given the distinct atherogenic profile of sdLDL particles, emerging therapeutic approaches are focusing on not just lowering overall LDL-C levels but also modifying the distribution of LDL subfractions. Interventions that increase the size of LDL particles, such as the use of fibrates or omega-3 FAs, have been shown to reduce cardiovascular risk, particularly in patients with a predominance of sdLDL (70). Similarly, lifestyle interventions such as dietary modifications and physical activity can significantly impact LDL particle size and density, highlighting the importance of a holistic approach to cardiovascular risk management (71).

Lp(a) levels, largely determined by genetics, show significant interindividual variability, ranging from <1 mg/dl to >1,000 mg/dl in the general population and remain stable throughout life. Elevated Lp(a) levels are linked to inflammatory conditions such as rheumatoid arthritis (RA) (72) and systemic lupus erythematosus (73). The atherogenic potential of Lp(a) is influenced by its structural properties and interactions with the vasculature. Lp(a) particles consist of an LDL core covalently bound to apo(a), which exhibits extensive size polymorphism. This variability arises from the number of kringle IV type 2 repeats in apo(a), affecting both particle size and plasma concentrations. Smaller apo(a) isoforms are associated with higher Lp(a) levels and increased atherogenicity. Lp(a) promotes atherogenesis through mechanisms including direct deposition onto arterial walls, facilitated by its greater susceptibility to oxidation compared to LDL (74). In fact, Lp(a) is the main transporter of oxPL in blood (75). Oxidized Lp(a) is readily taken up by macrophages via scavenger receptors, leading to foam cell formation, a key step in AS development. Additionally, elevated Lp(a) levels correlate inversely with vascular reactivity, contributing to endothelial dysfunction (76).

In addition to classifying LDL subfractions by size, density, or electronegativity, recent studies have emphasized the importance of quantifying particle mass composition, namely, the relative content of TG, free and esterified cholesterol, phospholipids, and protein within each subclass (77). These compositional profiles can vary significantly between LDL subfractions and are influenced by underlying metabolic states. For example, TG-enriched LDL particles are often found in insulin-resistant individuals and may correlate with delayed hepatic clearance and increased atherogenicity (78, 79). Advanced lipidomic approaches now allow for more precise characterization of these features, offering the potential to transform LDL subclass analysis from a descriptive tool into a quantitative, decision-support metric (80, 81). Incorporating compositional data may enhance cardiovascular risk stratification and support more individualized lipid-lowering strategies.

Electronegativity of LDL is a key factor in its atherogenic potential, with more electronegative LDL particles showing increased propensity for promoting AS. The formation of L5/LDL(-) occurs through various mechanisms, including oxidation, glycation, desialylation, and enzymatic modifications (82). These processes alter both the lipid and protein components of LDL, leading to changes in density, particle size, and surface charge. Oxidation, in particular, plays a crucial role in increasing LDL electronegativity, with oxPLs and modified apoB-100 contributing to the increased negative charge (83). Glycation of LDL, which is prevalent in diabetic conditions, also enhances L5/LDL(-)'s electronegativity and atherogenicity (40). Desialylation has also been related with the formation of L5/LDL(-) particles (84). Other factors, such as an abnormal conformation of apoB-100 (85, 86), increased apolipoprotein content (44, 45), and especially non-esterified fatty acids (NEFA) (87, 88), are also key determinants in increasing the electronegativity of LDL particles. Finally, the fact that a significant proportion of L5/LDL(-) is made up of sdLDL (88, 89) also contributes to an increase in electronegativity, since sdLDL has a greater negative charge than large or intermediate LDL particles (90).

The increased negative charge of LDL particles affects their interactions with extracellular matrix components and cell surface receptors. For instance, L5/LDL(-) shows a higher affinity for proteoglycans in the arterial wall, promoting its retention and accumulation in the subendothelial space (91, 92). Furthermore, L5/LDL(-) is preferentially recognized by scavenger receptors rather than the classical LDLR, leading to increased uptake by macrophages and foam cell formation (93). The electronegativity of LDL also influences its susceptibility to further modifications and aggregation, with more electronegative particles being more prone to additional oxidative changes and aggregation (94). Interestingly, recent studies have shown that L5/LDL(-) particles possess enzymatic activities, including platelet-activating factor acetyl hydrolase (95), phospholipase C (41) or ceramidase activities (36), which may play a role in modulating their effects on vascular cells (40). These properties contribute to the enhanced atherogenicity of L5/LDL(-). The presence of L5/LDL(-) in circulation has been associated with various pathological conditions, including CVD, DM, metabolic syndrome, and RA (96–102). Measurement of LDL electronegativity has been proposed as a potential biomarker for assessing cardiovascular risk, complementing traditional lipid profile analyses (83, 103, 104). Understanding the mechanisms underlying LDL electronegativity and its implications in atherogenesis provides valuable insights for developing targeted therapeutic strategies to mitigate the harmful effects of these modified lipoproteins in CVDs.

The complex interplay of genetic, metabolic, and lifestyle factors influences the physical and chemical properties of LDL particles such as density, size, composition, and oxidative state, which can have profound implications for their metabolism, clearance, and role in cardiovascular health (105, 106). These factors collectively determine the structural and functional characteristics of LDL, which in turn impact its behavior in lipid metabolism and its role in health and disease.

One key factor is genetic variation, particularly in genes related to apolipoproteins, lipid metabolism, and LDLR. For instance, variations in the APOB gene can alter the structure of apoB-100, which is the primary protein component of LDL, affecting its receptor-binding affinity and clearance rate (107). Similarly, polymorphisms in the LDLR gene can influence the uptake and degradation of LDL particles, leading to alterations in their plasma concentrations and composition (108). Genetic determinants also play a key role in Lp(a) atherogenicity. The LPA gene exhibits size polymorphisms due to a variable number of kringle IV type 2 repeats, which define the apo(a) isoform size. Fewer repeats result in smaller isoforms, which are linked to elevated plasma Lp(a) concentrations and a higher risk of AS and CVEs (109).

Metabolic conditions, such as insulin resistance and DM, also modulate LDL properties by altering lipid exchange and modifying enzymatic activities. Insulin resistance often leads to increased TG content in LDL, forming TG-enriched LDL particles, which are more prone to lipolysis by HL. This process generates sdLDL particles that are more atherogenic (106). Additionally, glycation of LDL particles in the context of hyperglycemia further modifies their structure, making them more susceptible to oxidative damage and less efficiently cleared by the LDLR pathway (110). These changes in LDL composition and size are further compounded by enzymatic activity involving LPL and CETP, which mediate lipid exchange among lipoproteins, altering the distribution of CEs and TGs across LDL, VLDL, and HDL fractions.

Lifestyle factors, such as diet, physical activity, and smoking, also play a significant role in shaping LDL particle properties. Diets high in saturated fats and cholesterol increase the hepatic production of apoB-containing lipoproteins, leading to elevated concentrations of large, buoyant LDL particles (111). Conversely, high-carbohydrate diets and excessive alcohol consumption tend to promote the formation of sdLDL particles by increasing hepatic VLDL production and enhancing the activity of CETP (105). Regular physical activity can reduce the concentration of sdLDL by improving insulin sensitivity and lowering TG levels, thus promoting the formation of larger, less dense LDL particles. On the other hand, smoking has been shown to increase oxidative stress, which oxidizes LDL lipids and proteins, making the particles more atherogenic and less recognizable by the LDLR (112).

The clinical relevance of LDL particle characteristics in atherogenicity has become a focal point in cardiovascular research, emphasizing the importance of not just LDL-C levels, but also the properties of LDL particles themselves. Atherogenesis is a disorder of the artery wall characterized by various stages: initial adhesion of monocytes and lymphocytes to the EC surface, subsequent migration of these cells into the sub-endothelial space, and, in the case of monocytes, differentiation into macrophages. The proatherogenic effect of these LDL subfractions is primarily due to their enhanced arterial wall penetration, increased susceptibility to oxidative or enzymatic modification, and greater uptake by macrophages through scavenger receptors, which promotes foam cell formation (113). This leads to the accumulation of CEs and the formation of foam cells, which, along with T lymphocytes, contribute to the development of atheroma plaque (Figure 1). Additionally, vascular SMCs migrate from the media into the intima and proliferate, resulting in the formation of atherosclerotic plaques (31). The entire atherogenic process involves crucial cellular activities like adhesion, migration, differentiation, proliferation, and interactions with the extracellular matrix (114–117).

Therapeutic approaches targeting LDL particle characteristics have evolved significantly in recent years, driven by a growing understanding of the distinct atherogenic potential associated with different LDL subfractions. Traditional lipid-lowering strategies have primarily focused on reducing overall LDL-C levels; however, emerging evidence highlights the necessity of addressing the qualitative characteristics of LDL particles, such as their density, size, and oxidative state (Table 2), to more effectively mitigate cardiovascular risk (Figure 2) (154).

The most established therapeutic strategy for lowering LDL-C is the use of statins, which inhibit the enzyme HMG-CoA reductase to reduce cholesterol synthesis in the liver. Statins not only lower overall LDL-C levels but have also been shown to shift the LDL particle distribution toward larger, more buoyant particles, which are considered less atherogenic (155). By doing so, statins can significantly reduce the incidence of CVEs. However, while statins remain the cornerstone of lipid management, their impact on LDL particle size varies among individuals, and not all patients achieve adequate LDL particle modification with statin therapy alone (62). To address the limitations of statin therapy, additional agents to modify lipid levels have been introduced to target specific LDL particle traits. For example, ezetimibe, a cholesterol absorption inhibitor, has been shown to further reduce LDL-C levels when used in conjunction with statins (154). Clinical trials indicate that this combination therapy can lead to a more pronounced decrease in sdLDL particles, potentially enhancing cardiovascular protection (25). In addition, it has been shown that therapies using different statins also decrease the proportion of L5/LDL(-) within total LDL (156–159).

In the context of sdLDL, novel and promising therapies aimed at reducing hypertriglyceridemia, such as inhibitors of the action of apoC-III or angiopoietin-like protein 3, including monoclonal antibodies, antisense oligonucleotides, or small interfering RNA (siRNA), are relevant since lowering TGs is expected to increase LDL particle size (Table 2; Figure 2).

Furthermore, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have emerged as a novel class of lipid-lowering agents that can dramatically lower LDL-C levels and alter LDL particle composition. PCSK9 inhibitors increase the number of available LDLRs on hepatocytes, promoting the clearance of atherogenic LDL particles from the circulation and thereby decreasing the concentration of sdLDL (160). Recent research has also investigated the potential of novel therapies such as apoB-targeting agents and antisense oligonucleotides to further refine LDL particle traits. These therapies aim to specifically reduce the concentration of apoB-containing lipoproteins, which include LDL and VLDL. Preliminary studies suggest that targeting apoB may lead to significant reductions in both LDL-C and the number of Lp(a) particles, thus lowering atherogenic risk (161). Olpasiran, a siRNA, blocks Lp(a) production by preventing translation of apo(a) mRNA (162, 163). Treatment with the oxLDL-specific antibody orticumab reduced aortic AS by 43%, subvalvular plaque area by 50% and the macrophage content by 31% (164).

In addition to pharmacological therapies, lifestyle modifications play a crucial role in targeting LDL particle traits. Diet, physical activity, and weight management can significantly influence LDL particle size and density (165, 166). Diets rich in omega-3 FAs, soluble fiber, and monounsaturated fats have been shown to favorably modify LDL particle characteristics by promoting larger, less atherogenic particles (167). Regular physical activity not only aids in weight loss but also enhances the lipid profile by increasing the proportion of large, buoyant LDL particles while reducing sdLDL particles (168).

Breakthroughs in personalized medicine and next-generation lipid analysis tools are revolutionizing therapeutic approaches through tailored strategies based on a patient's distinct LDL profile. By utilizing comprehensive lipid profiles, clinicians can identify patients with a predominance of sdLDL particles and implement targeted interventions to address these specific traits effectively (113).

Essentially, the landscape of therapeutic approaches targeting LDL particle traits has expanded significantly in recent years, incorporating a combination of pharmacological interventions, lifestyle modifications, and personalized medicine strategies. By focusing on not only the reduction of LDL-C but also the qualitative characteristics of LDL particles, healthcare providers can enhance the efficacy of cardiovascular risk management and potentially improve patient outcomes in the long term.

Emerging research efforts to understand the atherogenic contributions of LDL particles are increasingly focused on the complex mechanisms that underlie LDL's role in CVD. Traditional perspectives have primarily emphasized the quantity of circulating LDL-C as a risk factor; however, contemporary investigations are shifting toward a more nuanced understanding of LDL particle characteristics, including density, size, and biochemical modifications, as critical determinants of atherogenic potential. Recent studies have begun to elucidate how these LDL particle traits interact with various biological pathways, influencing the pathogenesis of AS and associated CVEs.

One promising area of research involves the exploration of LDL particle heterogeneity and its implications for vascular inflammation. New approaches utilizing advanced lipidomic profiling techniques aim to quantify and characterize different LDL subfractions in various populations and their respective roles in atherogenesis. By employing NMR spectroscopy and mass spectrometry, researchers can identify distinct LDL subclasses and their relative contributions to cardiovascular risk. This level of understanding could inform strategies that specifically counteract the atherogenic effects of distinct LDL subfractions (113).

Another emerging direction is the investigation of genetic and epigenetic factors that influence LDL particle characteristics. The role of genetic variants, such as those affecting apoB and PCSK9, in modulating LDL particle size and number is actively studied (1). Epigenetic modifications, such as DNA methylation and histone modifications, are also being investigated for their potential to influence LDL metabolism and particle composition, offering new avenues for therapeutic intervention (169). Understanding the genetic underpinnings of LDL particle traits could provide critical insights into individual susceptibility to atherogenicity and may inform personalized approaches to lipid management.

The role of gut microbiota in modulating LDL particle characteristics and their subsequent atherogenic potential represents another exciting research frontier. Emerging evidence suggests that the composition of gut microbiota can impact lipid metabolism, leading to alterations in LDL particle traits (170). Certain gut bacteria have been shown to influence the absorption and processing of dietary lipids, which may subsequently affect LDL particle formation and composition (171). Investigating the microbiome's impact on LDL particle characteristics could unveil novel therapeutic strategies that harness the gut-lipid axis to mitigate cardiovascular risk.

Additionally, the intersection of inflammation, oxidative stress, and LDL particle dynamics is garnering increasing attention. Recent studies have highlighted the role of inflammatory cytokines in modulating LDL particle composition, leading to a greater proportion of sdLDL particles (172). Understanding how systemic inflammation interacts with LDL metabolism may yield insights into the development of new anti-inflammatory therapies aimed at reducing LDL-related atherogenicity. This focus on inflammation aligns with the broader recognition of CVD as an inflammatory condition, necessitating a holistic approach to prevention and treatment.

Finally, the integration of novel imaging techniques, such as intravascular ultrasound and positron emission tomography, is enhancing our ability to visualize LDL particle behavior within the arterial wall and its relationship to plaque formation and stability (173). These technologies can provide real-time assessments of LDL dynamics and their contributions to atherogenesis, preparing the path for more precise risk stratification and management strategies (174). Emerging research directions in understanding LDL particle contributions to atherogenicity are multifaceted, focusing on the intricate interplay of particle characteristics, genetic and epigenetic influences, gut microbiota interactions, inflammation, and advanced imaging technologies. These innovative approaches hold the potential to deepen our understanding of LDL's role in CVD and inform the development of more effective, personalized therapeutic interventions.

Among atherogenic LDL subfractions, L5/LDL(-) has gained increasing attention due to its pro-inflammatory, endothelial-damaging, and atherogenic properties demonstrated in experimental settings (31). However, despite compelling in vitro and animal model data, the clinical evidence supporting L5/LDL(-) as a diagnostic or prognostic biomarker remains limited and preliminary. To date, the majority of L5/LDL(-) studies have been conducted in relatively small cohorts, often in highly selected populations such as patients with metabolic syndrome, T2DM, STEMI, or chronic autoimmune disorders (32, 89, 96, 99, 100, 152, 153, 159, 175–178). These studies, while suggestive, often lack sufficient power to generalize findings across broader populations. Furthermore, differences in lipoprotein separation techniques, L5/LDL(-) quantification methods, and patient phenotyping limit the reproducibility and comparability of findings across studies. Longitudinal data on L5/LDL(-) levels and cardiovascular outcomes are particularly scarce.

Moreover, many studies have not fully controlled for potential confounding factors such as concurrent lipid-lowering therapies, inflammation, or comorbid conditions that may influence both LDL electronegativity and cardiovascular risk. Importantly, while the association between elevated L5/LDL(-) levels and endothelial dysfunction has been shown mechanistically in vitro, direct causal links in human populations remain to be established.

Given these limitations, the role of L5/LDL(-) as a clinical biomarker must be interpreted with caution. The current level of evidence is best characterized as hypothesis-generating rather than conclusive. Future studies with larger, more diverse populations, standardized methods for L5/LDL(-) quantification, and longitudinal follow-up will be critical to validate L5/LDL(-)'s utility as a biomarker and therapeutic target in AS.

Current clinical practice guidelines, including those from the American College of Cardiology (ACC)/American Heart Association (AHA) and the European Society of Cardiology (ESC)/European Atherosclerosis Society (EAS), emphasize the central role of LDL-C in cardiovascular risk assessment and management (5, 179). Treatment strategies focus primarily on lowering total LDL-C through lifestyle modification and pharmacologic therapy, most notably statins, ezetimibe, and PCSK9 inhibitors, based on absolute risk categories and LDL-C thresholds.

Despite mounting evidence that certain LDL subfractions, such as sdLDL, oxLDL, Lp(a), and L5/LDL(-), may be more directly implicated in atherogenesis than total LDL-C, these subfractions are not currently incorporated into routine risk stratification algorithms or treatment guidelines (31). Several reasons underlie this gap, including the lack of standardized, widely available assays for subfraction measurement, limited large-scale clinical data linking subfractions to outcomes independently of LDL-C, and the absence of intervention studies specifically targeting these subfractions (9, 180).

Nevertheless, a growing body of research suggests that patients with normal or mildly elevated LDL-C levels may still carry a high burden of atherogenic LDL subfractions, highlighting the need for more refined lipid profiling in selected populations, such as those with metabolic syndrome, diabetes, or residual cardiovascular risk despite statin therapy (181). Emerging strategies, including lifestyle interventions, niacin, fibrates, and some newer lipid-modifying agents (e.g., angiopoietin-like protein 3 inhibitors), may preferentially impact these atherogenic subfractions, although their clinical roles remain to be fully defined (65, 182, 183).

As such, LDL subfraction analysis currently resides in the realm of research or specialized lipid clinics rather than mainstream clinical practice. Future inclusion in guidelines will likely depend on validation from large-scale, prospective studies demonstrating incremental predictive value and clear benefit from subfraction-targeted therapies.

This review comprehensively explores the multifaceted nature of atherogenic LDL particles by examining their density, size and electronegativity, and elucidates how these characteristics contribute to their atherogenic potential. Current classification systems provide a framework for distinguishing various LDL subfractions based on their physicochemical properties, yet understanding the underlying mechanisms requires robust methodological approaches for precise measurement and characterization. The evidence indicates that smaller, denser, and more electronegative LDL particles exhibit a higher propensity for contributing to atherogenesis due to enhanced susceptibility to oxidative modification and preferential uptake by macrophages.

Factors such as diet, genetics, and metabolic conditions further modulate LDL particle properties, adding another layer of complexity to their role in CVD risk. The clinical relevance of LDL subfractions and their distinct characteristics suggest that tailored therapeutic strategies targeting these properties may offer a more effective approach to reducing atherogenicity and mitigating CVD risk. This growing body of evidence underscores the importance of integrating these insights into clinical practice to improve risk stratification and therapeutic interventions.