The recent study by Martínez-Beamonte et al. provides new insights into how extra virgin olive oil enriched in bioactive compounds may exert anti-atherogenic effects, by improving high-density lipoprotein (HDL) function, as determined by the high-density lipoprotein-specific phospholipid efflux (HDL-SPE) assay in Apoe-deficient mice. We would like to highlight two points. First, the HDL-SPE assay was validated in human samples, but there is only limited data on how it performs in mice. Second, the role of ApoE in ApoA-I-containing HDL may differ substantially between mice and humans. The finding by Martínez-Beamonte et al. is quite intriguing, but it will be important to extend their findings in mice to human subjects.

The HDL-specific phospholipid efflux (HDL-SPE) assay is a novel cell-free measure of HDL function that is inversely associated with coronary artery disease. However, the effect of exercise training on HDL-SPE is unknown. The purpose of this study was to examine the effect of exercise training on HDL-SPE in a large, diverse cohort free of overt disease. Clinical and functional measures of HDL were taken before and after 20 weeks of endurance exercise training in 508 participants from the HERITAGE Family Study. Associations of HDL-SPE with HDL-related traits were examined using Pearson's correlations at baseline and following exercise training (significance: P < 7.4 × 10−4). The effect of exercise training on HDL-SPE was examined using paired t-tests (significance: P < 0.05). Mean (SD) HDL-SPE was 1.40 (0.19) and higher in females compared with males and in White participants compared with Black participants. Baseline HDL-SPE was strongly associated with HDL-C (r = 0.45) and apoA-I (r = 0.43, both P < 6.9 × 10−24) but not with measures of cholesterol efflux. Mean HDL-SPE increased (0.023, P = 0.002) following exercise training, but these increases only occurred in those with the lowest baseline HDL-SPE levels. Change in HDL-SPE was associated with changes in HDL-C (r = 0.27), medium HDL concentration (r = 0.24), and apoA-I and HDL size (r = 0.17, all P < 1.3 ×10−4). HDL-SPE increased following regular exercise, and changes in HDL-SPE were related to changes in HDL size and subclass concentrations. Our findings demonstrate that individuals at higher risk for coronary artery disease may experience the largest benefits from exercise training as related to this novel biomarker of HDL function.

This review describes the recently developed equations for calculating Low-density lipoprotein cholesterol (LDL-C), and equations for estimating small dense LDL-cholesterol (sdLDL-C), and LDL-triglycerides (LDL-TG) for atherosclerotic cardiovascular disease (ASCVD) risk assessment. The new Modified Sampson-NIH equation provides a more accurate estimation of LDL-C across a wide range of TG levels compared to the traditional and still commonly used Friedewald equation. Furthermore, it is more accurate compared to other equations at the low LDL-C cutpoints used for high-risk and very high-risk ASCVD patients and is valuable for deciding the need for additional lipid-lowering therapy. New equations for calculating sdLDL-C and LDL-TG use the same lipid parameters as for calculating LDL-C but offer additional insights into atherogenic lipoprotein burden. High plasma TG and very low LDL-C concentrations necessitate more accurate LDL-C calculations, which can be readily adopted without additional cost to improve ASCVD risk management.

Controlling hyperlipidemia has reduced but not eliminated atherosclerotic cardiovascular disease as a predominant cause of human mortality. Here, we report that loss of the immunoregulatory gene GTPase of immunity-associated protein 6 ( GIMAP6 ), causes an inflammatory vasculopathy and accelerated atherosclerosis in the absence of hyperlipidemia. These pathologic changes in turn result in progressive cardiac ischemia, myocardial infarction, and heart failure, culminating in early death. In humans, rare deleterious GIMAP6 variants are associated with premature severe cardiovascular disease. These findings reveal GIMAP6 to play an important protective role against atherosclerotic cardiovascular disease whose identification offers opportunities for improved risk management and a target for new therapies.

Accurate assessment of low-density lipoprotein cholesterol (LDL-C) is central to the prevention and treatment of atherosclerotic cardiovascular disease (ASCVD). Recent clinical guidelines have emphasized lower LDL-C targets and the use of non-fasting lipid testing, increasing the need for more reliable LDL-C estimation methods than the traditional Friedewald calculation. The development of modern Martin and Sampson methods has led to improved accuracy across a wider range of LDL-C and triglyceride levels. The most recent Modified Sampson method further enhances performance, particularly at low LDL-C levels. These modern LDL-C methods have been incorporated into up-to-date cholesterol management guidelines, which now recommend modern LDL-C methods over the Friedewald calculation. This review summarizes the evolution of LDL-C testing method and its integration into clinical practice to support more precise ASCVD management.

BACKGROUND:Analytical performance specifications for laboratory tests are essential components of quality assurance for clinical laboratories. A widely adopted performance criterion is total allowable error (TEa), which includes contributions from both bias and imprecision, which may contribute to test inaccuracies. However, the relationship of TEa to test result misclassification, a clinically relevant quality measure, is unclear. METHODS:Hypothetical clinical laboratory test results for 4 test models were generated and subjected to proportional bias and imprecision. Test misclassification (TM) as a function of bias and imprecision was determined using pre-defined cutpoint(s). Simulation analyses were then performed on 14 chemistry analytes using 3 data sets from patient results reported at the National Institutes of Health (NIH). RESULTS:We observed a complex and nonlinear relationship between bias and imprecision, and their impact on TM was not additive as may have been expected with TEa. TM scores were influenced by population distribution, the location of cutpoints, and the fraction of abnormal test values at baseline. Stringent TEa requirements did not correspond to low TM scores. On the contrary, TM scores for electrolytes were among the highest. Instead, TM scores closely correlated with the ratio of TEa to the width of the population distribution. CONCLUSION:TM has the advantage of correctly accounting for the differential effects of bias and imprecision. Compared to TEa, it provides a more accessible metric for evaluating performance and the clinical impact of errors.

Apolipoprotein C2 (APOC2) has been identified as a potential therapeutic target for acute myeloid leukemia, but its impact on normal hematopoiesis remains unclear. Here we generated a global inducible Apoc2 knockout (KO) mouse model by breeding R26-CreERT2 mice with mice homozygous for Apoc2 lox sites (Apoc2 fl/fl) over three generations. Global Apoc2 KO was induced in 4-8-week-old mice by 5 consecutive days of intraperitoneal administration of tamoxifen. Successful Apoc2 KO was confirmed by measuring Apoc2 mRNA levels, showing 80-99% reduction in the liver, bone marrow, spleen and blood tissues compared with wild type (WT). Despite significantly higher serum triglyceride levels in the Apoc2-KO group, no apparent abnormal phenotypes were observed. In vitro expansion of the hematopoietic stem and progenitor cells and splenocytes from Apoc2-KO were comparable to that of WT mice. In addition, Apoc2 deletion did not significantly affect the colony-forming ability or the competitive repopulation ability of hematopoietic stem and progenitor cells. Hematological analysis revealed no significant differences between Apoc2-KO and WT mice. RNA-sequencing analysis of bone marrow samples from the Apoc2-KO group identified four significantly downregulated genes and five upregulated genes, including Gm16867, which is orthologous to the human SLC25A37 gene involved in mitochondrial iron homeostasis. Gene set enrichment analysis revealed the enrichment of heme metabolism (false discovery rate 0.077). Our study demonstrates that deletion of Apoc2 exhibits limited impact on normal hematopoiesis under normal conditions. Future characterization of this Cre-inducible Apoc2-KO mice under stress conditions or during aging is needed to fully demonstrate the biological impact of Apoc2 deletion.

Apolipoprotein AV (APOA5) regulates intravascular triglyceride metabolism by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its ability to unfold the native conformation of lipoprotein lipase (LPL). LPL unfolding results in loss of catalytic activity and the detachment of LPL from the surface of cells. An APOA5 truncation mutation (identified in two patients with hypertriglyceridemia) had suggested that the last 35 amino acids of APOA5 are important for its function. We reasoned that a monoclonal antibody (mAb) against carboxyl-terminal sequences in APOA5 could clarify functionally important amino acid residues in APOA5 and assist in elucidating the mechanism by which APOA5 regulates plasma triglyceride metabolism. Because carboxyl-terminal APOA5 sequences are evolutionarily conserved, we began by screening a human Fab bacteriophage library for binders of carboxyl-terminal APOA5 sequences. We identified one such binder and used phage DNA sequences to build a chimeric IgG1 mAb (IBA707) against APOA5. The binding of IBA707 to APOA5 was abolished by nonconservative amino acid substitutions in conserved sequences (residues L337-I348) within a C-terminal α-helix in APOA5. The same substitutions disrupted APOA5's ability to bind and inhibit ANGPTL3/8 activity. IBA707-mediated blockade of APOA5 function reduced intracapillary LPL levels and triggered elevated plasma levels of triglycerides and ANGPTL3/8 in both fasted and refed mice. IBA707 was cleared rapidly from the plasma in Apoa5+/+ mice but slowly in Apoa5-/- mice. Our studies identified functionally important amino acids in APOA5 and revealed that APOA5 controls plasma triglyceride metabolism in part by modulating plasma levels of ANGPTL3/8.

BACKGROUND Familial hypobetalipoproteinemia is a rare autosomal codominant disorder, often caused by a defect in apolipoprotein B (apoB) production required for lipoprotein formation and secretion. OBJECTIVE Characterization of the lipid profiles of 3 family members exhibiting very low circulating cholesterol levels. METHODS Plasma samples from the control sibling and the affected patients were analyzed. Fast protein liquid chromatography and high-performance liquid chromatography were used to characterize the lipid profiles, size, and distribution of lipoprotein particles. Exome sequencing of family members revealed a single-nucleotide deletion in APOB in the 3 affected individuals. The effect of the single-nucleotide deletion on the secretion of apoB was analyzed in Immortalized Human Hepatocyte (IHH) cells. RESULTS Plasma lipid profiles revealed that the affected individuals have low levels of total cholesterol and low-density lipoprotein cholesterol, with no difference in lipoprotein particle size. DNA sequencing of APOB revealed a single heterozygote deletion of an adenosine in exon 3 at the nucleotide position 1268 in all affected members. This deletion introduces a reading frame shift at glutamine 380, resulting in a stop codon at position 397. The C-terminally truncated apoB, called apoB9, is a variant spanning ∼9% of the full-length protein. Upon expression of apoB9 in IHH cells, the protein did not exit the endoplasmic reticulum/cis-Golgi and, hence, was not secreted into the media. Molecular modeling revealed that apoB9 lacks the βA- and βB-sheets that are required for lipid particle formation, which can explain the absence of apoB9 secretion. CONCLUSION Our data suggested that the affected family members have ∼50% to 60% lower apoB levels and are likely protected against the development of atherosclerosis and cardiovascular diseases.

Background:Familial dysbetalipoproteinemia (FDB) is a genetic lipoprotein disorder that can develop in patients homozygous for the APOE2 genotype (ε2/ε2). It is associated with decreased clearance of remnant lipoproteins and increased atherosclerotic cardiovascular disease (ASCVD) risk disproportionate to their level of LDL-C. A goal of this study was to develop a screening test for the ε2/ε2 genotype based on routinely available lipid tests and to determine those at most risk for ASCVD. Methods:After assembly of a primary prevention cohort from the UK Biobank (n= 269,895), gene array and exome data was utilized to classify patients as being ε2/ε2 genotype positive or negative. Lipid profiles and APOB levels were extracted and the number of ASCVD events was tabulated during a 15-year follow-up period. Results:Using a newly developed equation for estimating APOB (eAPOB) with lipid panel test results, the ratio of measured APOB to eAPOB was better than any other individual lipid test or ratio for identifying patients with the ε2/ε2 genotype (AUC: APOB/eAPOB: 0.990 (0.986-0.994), nonHDL-C/APOB: 0.961 (0.952-0.970), APOB: 0.955 (0.949-0.961), VLDL/TG: 0.788 (0.771-0.804)). The majority of ε2/ε2 patients could be identified with the APOB/eAPOB ratio even before they expressed the FDB phenotype with elevated TG and nonHDL-C. The PCE or PREVENT risk equations were the most accurate method for identifying higher risk patients (AUC: PREVENT: 0.690 (0.637-0.742), PCE: 0.697 (0.645-0.749)). Conclusion:The APOB/eAPOB ratio can be used to accurately identify the ε2/ε2 genotype and conventional risk equations are the best method for determining those at risk for ASCVD.

Accurate laboratory assessment of circulating lipids underpins cardiovascular risk stratification, yet clinical interpretation depends not only on the assays but on the formula chosen to estimate low-density lipoprotein cholesterol (LDL-C). This review integrates the 2019-2025 evidence on laboratory methods for triglycerides (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDLC), and on the formulas estimating LDL-C, VLDL-C, and non-HDL cholesterol, to determine how these should be measured, reported, and harmonized in Brazil, where lipid thresholds are adapted from international consensus. A PRISMA 2020 systematic search (PROSPERO CRD420251241064) of PubMed/MEDLINE, Scopus, SciELO, LILACS, Web of Science, and Embase retrieved 57,915 records; after removing 38,210 duplicates, 19,705 titles/abstracts were screened, 312 full texts assessed, and 25 sources included. Enzymatic colorimetric assays remain standard for TG, TC, and HDLC. For LDL-C, Martin/Hopkins classifies more accurately than Friedewald (89.6% vs 83.2% correct categorization in 5,051,467 patients), particularly at high TG and low LDL-C, while Sampson/NIH and modified Sampson/NIH extend reliable estimation into hypertriglyceridemia and very low LDL-C; direct measurement is reserved for TG beyond the validated range. Although the review centers on the Friedewald, Martin/Hopkins, and Sampson/NIH families that dominate guideline practice, other published equations exist and are addressed in context. In Brazil, atherogenic-lipid thresholds are risk-based decision limits rather than reference intervals; national surveys describe lipid distributions but were not designed to establish them. Analytical standardization through traceability programs, multicenter validation of formulas, and-where the distribution-based construct applies (HDLC, pediatrics)-nationally derived reference intervals are priorities for equitable cardiovascular risk assessment in Brazil.

Background: The biophysical regulation of HDL function is driven by its lipid composition. While research has transitioned from HDL-cholesterol to assessing HDL functionality, the molecular mechanisms modifying the directionality and stoichiometry of HDL phospholipid and cholesterol transfer in human plasma are poorly understood. Objectives: To determine how nanoparticle phospholipid acyl-chain composition controls the directionality, stoichiometry, and surface remodeling of HDL during free cholesterol (FC) exchange in human plasma. Methods and Results: We developed nanoparticles coated with phospholipids (PL) with varying acyl-chain length and saturation (DMPC, SOPC, DOPC, POPC, SAPC, EggPC) with matching molar PL content. Lipid exchange between nanoparticles containing either PL alone (acceptor particle) or equimolar PL:FC ratios (donor particle) was assayed in either whole or apoB-depleted plasma. Total plasma FC removal by acceptor nanoparticles was invariant across PL compositions (30-40%), suggesting a surface-limited exchange process dominated by apoB-containing lipoproteins. In marked contrast, HDL-specific FC exchange was highly sensitive to nanoparticle PL composition (22-66%). When nanoparticles acted as FC donors, unsaturated and polyunsaturated PL selectively transferred FC to HDL, whereas saturated or short-chain PL did so to a lesser extent. This directional FC delivery lowered HDL PL:FC ratios from 5.6 at baseline to 1.7-2.0, demonstrating a highly efficient FC uptake (47-94% increase). Conversely, when nanoparticles acted as acceptors, HDL PL:FC ratios increased markedly (from 5.6 up to 11-18), revealing net PL enrichment of HDL, despite FC loss. These HDL lipid stoichiometric shifts reveal a dynamic adaptive remodeling of both PL and FC on the HDL surface, which may be modulated by acyl-chain-dependent physical constraints. Conclusions: The directionality of plasma HDL-cholesterol transfer may be regulated by surface lipid dynamics. Phospholipid acyl-chain-dependent membrane lipid disorder selectively alters FC transferability, while HDL PL content dynamically remodels to preserve surface stability. HDL acts as a mechanically adaptive surface rather than a fixed cholesterol reservoir. Thus, the directionality of surface lipid exchange, PL:FC stoichiometry, and acyl-chain-dependent surface plasticity underlies HDL functionality.

Cardiovascular disease is a major cause of human morbidity and mortality. Drug strategies for the prevention of the disease are largely centered on the interaction of low-density lipoprotein receptor (LDLR) with the apolipoprotein B-100 (apoB-100) protein on low-density lipoprotein (LDL). Recently, the structure of apoB-100 on LDL was determined in the absence and presence of LDLR, using cryo-electron microscopy. A remarkable structural feature of apoB-100 is the lack of any significant tertiary structure within the C-terminal two-thirds of the protein (>3000 residues). Instead, apoB-100 forms amphipathic helices and β-sheets on the phospholipid surface of LDL, which envelops its neutral lipid core. The apoB-100 ligand binding domain for LDLR includes multiple points on a circumferential β-belt and on the N terminus. In the course of this study, we also observed several instances of structural heterogeneity in apoB-100. The various conformations may allow apoB-100 to accommodate different size lipoprotein particles and to permit recognition by other apolipoproteins or receptors.

CONTEXT:Combined oral contraceptives (OCPs) and metformin are commonly used in patients with polycystic ovary syndrome (PCOS), who are at elevated risk of dyslipidemia and cardiovascular disease (CVD), but their effects on advanced lipid phenotyping remain unclear. OBJECTIVE:To examine the impact of OCPs and metformin on (1) lipoproteins, (2) apolipoproteins, and (3) cholesterol efflux capacity (CEC, marker of HDL function). DESIGN:Secondary analysis of the COMET-PCOS randomized clinical trial. SETTING:Two tertiary care reproductive endocrinology clinics. PATIENTS OR OTHER PARTICIPANTS:Patients with hyperandrogenic PCOS and elevated BMI with paired serum samples for lipoprotein/apolipoprotein analysis (n = 180) and cholesterol efflux capacity (n = 129). INTERVENTION(S):24 weeks of metformin, OCP, or OCP + metformin. MAIN OUTCOME MEASURE(S):Change in HDL particles (HDL-P), low-density lipoprotein particles (LDL-P), triglyceride-rich lipoprotein particles (TRL-P); apolipoproteins A-I, B, and C-III; and CEC. RESULTS:HDL-P concentration increased in the OCP and OCP + metformin arms, while atherogenic small LDL-P increased slightly. Metformin had a largely neutral effect on advanced lipid phenotyping. Cholesterol efflux capacity increased in the OCP arm, though this was attenuated when adjusting for change in ApoA-I (adjusted ratio of geometric means 1.08, 95% CI 0.99-1.17), which increased in all arms. CONCLUSIONS:In patients with PCOS and high metabolic risk, OCP use resulted in improved HDL-C function and a mixed effect on lipoproteins and apolipoproteins, with no clear benefit from metformin. These findings do not support or refute the cardiovascular risk-benefit of OCP use in PCOS and highlight the need for studies evaluating long-term clinical outcomes.

Background Heart failure (HF) is a complex syndrome with high mortality. The Metabolic Vulnerability Index (MVX) is a novel multimarker score derived from inflammation and malnutrition markers that has demonstrated prognostic value in patients with HF; however, changes in MVX over time have not been characterized. The purpose of this study was to evaluate change between baseline and 12-month measurements of the MVX and its components in an HF clinical trial population.Methods Nuclear magnetic resonance spectroscopy (NMR) MetaboProfile analyses (Labcorp) were performed on paired EDTA baseline and 12-month plasma samples collected from 46 HF patients with preserved ejection fraction (HFpEF) enrolled in the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial (TOPCAT) clinical trial to generate non-sex-specific MVX scores and component analytes. Paired t-tests were used to test the null hypothesis that the differences in paired population means between baseline and 12-months for MVX, or its components, were equal to 0. In sensitivity analyses, we evaluated whether differences in MVX or component values differed by drug status.Results In this sample, the mean (SD) age was 72.5 (9.2) years; 50% were female, and 89% were White. The prevalence of comorbidities was similar to the full TOPCAT population. MVX mean scores were not significantly different between baseline and 12-month measurements (mean difference = 1.52, 95% CI: -0.97 to 4.01, P = 0.225).Conclusions MVX scores appeared stable in this cohort over one year. Future studies should evaluate changes in serial measurements of MVX in a larger sample size.

Plasma levels of high-density lipoprotein (HDL)-cholesterol (HDL-C) is currently a key metric for the clinical assessment of cardiovascular disease (CVD) risk. HDL-mediated removal of plaque lipids from cells and extracellular deposits in the arterial wall is one of several anti-atherogenic functions of HDL that may account for its inverse association with CVD risk. The HDL-mediated removal of cellular cholesterol in vitro has recently proven to be an even better predictor of CVD risk than HDL-C. HDL is composed of a heterogeneous population of particles, which perform different functions. HDL particles involved in the removal of atherosclerotic plaque lipids contain exchangeable apolipoproteins, mostly apolipoprotein A-I, that dissociate from the particle and then remove plaque lipids. Nascent HDL is formed by the solubilization and removal of both phospholipids and cholesterol from ABCA1-generated cellular plasma membrane domains by HDL-derived exchangeable apolipoproteins. This process plays a critical role in both the prevention and regression of atherosclerotic plaque. Herein, we describe the protocol for the cell-free, HDL-specific phospholipid efflux (HDL-SPE) assay, which we have previously shown can predict incident CVD risk. This assay specifically measures HDL apolipoprotein-mediated removal of a non-exchangeable fluorescent phospholipid from a lipid donor particle. Our case-control clinical studies have established that the HDL-SPE assay performs as well as and potentially even better than the "gold-standard" cell-based cholesterol efflux capacity assay, a laborious and technically complex assay that often utilizes radioactive cholesterol. The goal of this protocol is to enable basic and clinical researchers alike to assess the functionality of HDL in lipid mobilization, using a simple, standardized, cell-free, high-throughput assay. This assay can be used by basic researchers to gain insights into mechanisms underlying HDL-mediated lipid efflux and for screening new therapeutic agents that enhance HDL functionality. HDL-SPE can also serve as a clinical laboratory diagnostic assay for CVD risk assessment.