Background and aims: Low-density lipoprotein (LDL) plasma concentration decline is a biomarker for acute inflammatory diseases, including coronavirus disease-2019 (COVID-19). Phenotypic changes in LDL during COVID-19 may be equally related to adverse clinical outcomes.Methods: Individuals hospitalized due to COVID-19 (n = 40) were enrolled. Blood samples were collected on days 0, 2, 4, 6, and 30 (D0, D2, D4, D6, and D30). Oxidized LDL (ox-LDL), and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity were measured. Ina consecutive series of cases (n = 13), LDL was isolated by gradient ultracentrifugation from D0 and D6 and was quantified by lipidomic analysis. Association between clinical outcomes and LDL phenotypic changes was investigated.Results: In the first 30 days, 42.5% of participants died due to Covid-19. The serum ox-LDL increased from D0 to D6 (p < 0.005) and decreased at D30. Moreover, individuals who had an ox-LDL increase from D0 to D6 to over the 90th percentile died. The plasma Lp-PLA2 activity also increased progressively from D0 to D30 (p < 0.005), and the change from D0 to D6 in Lp-PLA2 and ox-LDL were positively correlated (r = 0.65, p < 0.0001). An exploratory untargeted lipidomic analysis uncovered 308 individual lipids in isolated LDL particles. Paired-test analysis from D0 and D6 revealed higher concentrations of 32 lipid species during disease progression, mainly represented by lysophosphatidyl choline and phosphatidylinositol. In addition, 69 lipid species were exclusively modulated in the LDL particles from non-survivors as compared to survivors.Conclusions: Phenotypic changes in LDL particles are associated with disease progression and adverse clinical outcomes in COVID-19 patients and could serve as a potential prognostic biomarker.(c) 2023 Published by Elsevier Inc.

Background Blockade of the lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) is a potentially attractive mechanism for lowering inflammatory and lipid risk in patients with atherosclerosis. This study aims to assess the safety, tolerability, and target engagement of MEDI6570, a high‐affinity monoclonal blocking antibody to LOX‐1. Methods and Results This phase 1, first‐in‐human, placebo‐controlled study (NCT03654313) randomized 88 patients with type 2 diabetes to receive single ascending doses (10, 30, 90, 250, or 500 mg) or multiple ascending doses (90, 150, or 250 mg once monthly for 3 months) of MEDI6570 or placebo. Primary end point was safety; secondary and exploratory end points included pharmacokinetics, immunogenicity, free soluble LOX‐1 levels, and change in coronary plaque volume. Mean age was 57.6/58.1 years in the single ascending doses/multiple ascending doses groups, 31.3%/62.5% were female, and mean type 2 diabetes duration was 9.7/8.7 years. Incidence of adverse events was similar among cohorts. MEDI6570 exhibited nonlinear pharmacokinetics, with terminal half‐life increasing from 4.6 days (30 mg) to 11.2 days (500 mg), consistent with target‐mediated drug disposition. Dose‐dependent reductions in mean soluble LOX‐1 levels from baseline were observed (>66% at 4 weeks and 71.61–82.96% at 10 weeks in the single ascending doses and multiple ascending doses groups, respectively). After 3 doses, MEDI6570 was associated with nonsignificant regression of noncalcified plaque volume versus placebo (−13.45 mm3 versus −8.25 mm3). Conclusions MEDI6570 was well tolerated and demonstrated dose‐dependent soluble LOX‐1 suppression and a pharmacokinetic profile consistent with once‐monthly dosing. Registration URL: https://clinicaltrials.gov/; Unique identifier: NCT03654313.

This is the source dataset for the publication of the same title in Communications Medicine

Background Psoriasis is a chronic inflammatory condition associated with coronary artery disease risk. Uptake of oxidized low‐density lipoprotein by the lectin‐like low‐density lipoprotein receptor‐1 triggers release of the soluble extracellular domain of the receptor (sLOX‐1). We sought to characterize the relationship between sLOX‐1, inflammation, and coronary plaque progression in psoriasis. Methods and Results A total of 327 patients with psoriasis had serum sLOX‐1 levels measured at baseline by an ELISA‐based assay. Stratification by high‐sensitivity C‐reactive protein ≥4.0 mg/L (quartile 4), identified 81 participants who had coronary plaque phenotyping at baseline and were followed longitudinally by coronary computed tomography angiography. Subjects within high‐sensitivity C‐reactive protein quartile 4 were middle‐aged (51.47±12.62 years), predominantly men (54.3%) with moderate psoriasis disease severity (6.60 [interquartile range, 3.30–13.40]). In the study cohort, participants with sLOX‐1 above the median displayed increased vulnerable coronary plaque features. At baseline, sLOX‐1 was associated with total burden (rho=0.296; P=0.01), noncalcified burden (rho=0.286; P=0.02), fibro‐fatty burden (rho=0.346; P=0.004), and necrotic burden (rho=0.394; P=0.002). A strong relationship between sLOX‐1, noncalcified burden (β=0.19; P=0.03), and fibro‐fatty burden (β=0.29; P=0.003) was found in fully adjusted models at baseline and 1‐ and 4‐year follow‐up. Finally, coronary plaque features progressed over 1 year regardless of biologic or systemic treatment in subjects with high sLOX‐1. Conclusions Patients with psoriasis with both high sLOX‐1 and high‐sensitivity C‐reactive protein levels have increased coronary plaque burden associated with atherosclerotic plaque progression independent of biologic and systemic treatment. Thus, sLOX‐1 might be considered as a promising marker in coronary artery disease risk estimation beyond traditional risk factors. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01778569.

Lipoprotein-X (LpX) are abnormal nephrotoxic lipoprotein particles enriched in free cholesterol and phospholipids. LpX with distinctive lipid compositions are formed in patients afflicted with either familial LCAT deficiency (FLD) or biliary cholestasis. LpX is difficult to detect by standard lipid stains due to the absence of a neutral lipid core and because it is unstable upon storage, particularly when frozen. We have recently reported that free cholesterol-specific filipin staining after agarose gel electrophoresis sensitively detects LpX in fresh human plasma. Herein, we describe an even more simplified qualitative method to detect LpX in both fresh and frozen–thawed human FLD or cholestatic plasma. Fluorescent cholesterol complexed to fatty-acid-free BSA was used to label LpX and was added together with trehalose in order to cryopreserve plasma LpX. The fluorescent cholesterol bound to LpX was observed with high sensitivity after separation from other lipoproteins by agarose gel electrophoresis. This methodology can be readily developed into a simple assay for the clinical diagnosis of FLD and biliary liver disease and to monitor the efficacy of treatments intended to reduce plasma LpX in these disease states.

Aims Reverse cholesterol transport (RCT) removes cholesterol and stabilizes vulnerable plaques. In addition, high-density lipoprotein (HDL) may be cardioprotective in acute myocardial infarction (MI). Lecithin-cholesterol acyltransferase (LCAT) may enhance RCT. The objective of this study was to investigate the pharmacokinetics, pharmacodynamics, and safety of multiple ascending doses of recombinant human LCAT (MEDI6012) to inform a Phase 2b programme. Methods and results This was a randomized, blinded, placebo-controlled, dose-escalation Phase 2a study of MEDI6012. Patients were randomized into one of four cohorts (40, 120, 300 mg IV weekly x3 doses, or 300 mg IV-push, 150 mg at 48 h and 100 mg at 7 days). All cohorts were planned to randomize 6:2 (MEDI6012 vs. placebo). The primary endpoints were baseline-adjusted area under the curve (AUC) from 0 to 96 h post dose 3 (AUC 0-96 h) for HDL-C, HDL cholesteryl ester (HDL-CE), and total cholesteryl ester (CE). The primary safety endpoints were treatment-emergent adverse events. A total of 32 patients were randomized. MEDI6012 significantly increased AUC 0-96 h for HDL-C, HDL-CE and CE in a graded fashion with increasing doses. Relative to placebo, MEDI6012 increased HDL-C at Day 19 by 66% (95% CI 33-99, P = 0.014) with 120 mg and 144% (95% CI 108-181, P < 0.001) with 300 mg. An IV-push increased HDL-C by 40.8% at 30 min. Overall adverse events were similar between groups with no severe, life-threatening/fatal adverse events, or neutralizing antibodies. Conclusions Multiple ascending doses of MEDI6012 were safe and well tolerated and significantly increased HDL-C, HDL-CE and CE in a dose-related manner. These data support the ongoing Phase 2b programme investigating MEDI6012 in ST-elevation MI.

BACKGROUND Dyslipoproteinemias can be classified by their distinct lipoprotein patterns, which helps determine atherosclerotic cardiovascular disease (ASCVD) risk and directs lipid management but this has required advanced laboratory testing. OBJECTIVE To develop a new algorithm for classifying lipoprotein disorders that only relies on the standard lipid panel. METHODS Lipid thresholds for defining the different lipoprotein phenotypes were derived for Non-High-Density Lipoprotein-Cholesterol (NonHDL-C) and Triglycerides (TG) to be concordant when possible with the current US Multi-Society guidelines for blood cholesterol management. RESULTS The new classification method categorizes patients into all the classical Fredrickson-like phenotypes except for Type III dysbetalipoproteinemia. In addition, a new hypolipidemic phenotype (Type VI) due to genetic mutations in apoB-metabolism is described. The validity of the new algorithm was confirmed by lipid analysis by NMR (N = 11,365) and by concordance with classification by agarose gel electrophoresis/beta-quantification (N = 5504). Furthermore, based on the Atherosclerosis Risk in Communities (ARIC) cohort (N = 14742), the lipoprotein phenotypes differ in their association with ASCVD (TypeV > IIb > IVb > IIa > IVa > normolipidemic) and can be used prognostically as risk enhancer conditions in the management of patients. CONCLUSIONS We describe a clinically useful lipoprotein phenotyping system that is only dependent upon the standard lipid panel. It, therefore, can be easily implemented for increasing compliance with current guidelines and for improving the care of patients at risk for ASCVD.

Background MEDI6012 is recombinant human lecithin cholesterol acyltransferase, the rate‐limiting enzyme in reverse cholesterol transport. Infusions of lecithin cholesterol acyltransferase have the potential to enhance reverse cholesterol transport and benefit patients with coronary heart disease. The purpose of this study was to test the safety, pharmacokinetic, and pharmacodynamic profile of MEDI6012. Methods and Results This phase 2a double‐blind study randomized 48 subjects with stable coronary heart disease on a statin to a single dose of MEDI6012 or placebo (6:2) (NCT02601560) with ascending doses administered intravenously (24, 80, 240, and 800 mg) and subcutaneously (80 and 600 mg). MEDI6012 demonstrated rates of treatment‐emergent adverse events that were similar to those of placebo. Dose‐dependent increases in high‐density lipoprotein cholesterol were observed with area under the concentration‐time curves from 0 to 96 hours of 728, 1640, 3035, and 5318 should be: mg·h/mL in the intravenous dose groups and 422 and 2845 mg·h/mL in the subcutaneous dose groups. Peak mean high‐density lipoprotein cholesterol percent change was 31.4%, 71.4%, 125%, and 177.8% in the intravenous dose groups and 18.3% and 111.2% in the subcutaneous dose groups, and was accompanied by increases in endogenous apoA1 (apolipoprotein A1) and non‐ATP‐binding cassette transporter A1 cholesterol efflux capacity. Decreases in apoB (apolipoprotein B) were observed across all dose levels and decreases in atherogenic small low‐density lipoprotein particles by 41%, 88%, and 79% at the 80‐, 240‐, and 800‐mg IV doses, respectively. Conclusions MEDI6012 demonstrated an acceptable safety profile and increased high‐density lipoprotein cholesterol, endogenous apoA1, and non‐ATP‐binding cassette transporter A1 cholesterol efflux capacity while reducing the number of atherogenic low‐density lipoprotein particles. These findings are supportive of enhanced reverse cholesterol transport and a functional high‐density lipoprotein phenotype. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02601560.

Objective: Functional HDL (high-density lipoprotein) particles that facilitate cholesterol efflux may be cardioprotective. EL (endothelial lipase) hydrolyzes phospholipids promoting catabolism of HDL and subsequent renal excretion. MEDI5884 is a selective, humanized, monoclonal, EL-neutralizing antibody. We sought to determine the safety, pharmacokinetics, and pharmacodynamic effects of multiple doses of MEDI5884 in patients with stable coronary artery disease. Approach and Results: LEGACY was a phase 2a, double-blind, placebo-controlled, parallel-design trial that randomized 132 patients with stable coronary artery disease receiving high-intensity statin therapy to 3 monthly doses of 1 of 5 dose levels of MEDI5884 (50, 100, 200, 350, or 500 mg SC) or matching placebo. The primary end point was the safety and tolerability of MEDI5884 through the end of the study (day 151). Additional end points included change in HDL cholesterol and cholesterol efflux from baseline to day 91, hepatic uptake of cholesterol at day 91, changes in various other lipid parameters. The incidence of adverse events was similar between the placebo and MEDI5884 groups. In a dose-dependent manner, MEDI5884 increased HDL cholesterol up to 51.4% ( P <0.0001) and global cholesterol efflux up to 26.2% ([95% CI, 14.3–38.0] P <0.0001). MEDI5884 increased HDL particle number up to 14.4%. At the highest dose tested, an increase in LDL (low-density lipoprotein) cholesterol up to 28.7% ( P <0.0001) and apoB (apolipoprotein B) up to 13.1% ( P =0.04) was observed with MEDI5884. However, at the potential target doses for future studies, there was no meaningful increase in LDL cholesterol or apoB. Conclusions: Inhibition of EL by MEDI5884 increases the quantity and quality of functional HDL in patients with stable coronary artery disease on high-intensity statin therapy without an adverse safety signal at the likely dose to be used. These data support further clinical investigation. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03351738.

Cardiovascular disease ( CVD) is the leading global cause of death, and treatments that further reduce CV risk remain an unmet medical need. Epidemiological studies have consistently identified low high-density lipoprotein cholesterol (HDL-C) as an independent risk factor for CVD, making HDL elevation a potential clinical target for improved CVD resolution. Endothelial lipase (EL) is a circulating enzyme that regulates HDL turnover by hydrolyzing HDL phospholipids and driving HDL particle clearance. Using MEDI5884, a first-in-class, EL-neutralizing, monoclonal antibody, we tested the hypothesis that pharmacological inhibition of EL would increase HDL-C by enhancing HDL stability. In nonhuman primates, MEDI5884 treatment resulted in lasting, dose-dependent elevations in HDL-C and circulating phospholipids, confirming the mechanism of EL action. We then showed that a favorable lipoprotein profile of elevated HDL-C and reduced low-density lipoprotein cholesterol (LDL-C) could be achieved by combining MEDI5884 with a PCSK9 inhibitor. Last, when tested in healthy human volunteers, MEDI5884 not only raised HDL-C but also increased HDL particle numbers and average HDL size while enhancing HDL functionality, reinforcing EL neutralization as a viable clinical approach aimed at reducing CV risk.

Atherosclerosis, the underlying cause of cardiovascular disease (CVD), is a worldwide cause of morbidity and mortality. Reducing ApoB-containing lipoproteins—chiefly, LDL (low-density lipoprotein)—has been the main strategy for reducing CVD risk. Although supported by large randomized clinical trials, the persistence of residual cardiovascular risk after effective LDL reduction has sparked an intense search for other novel CVD biomarkers and therapeutic targets. Recently, Lox-1 (lectin-type oxidized LDL receptor 1), an innate immune scavenger receptor, has emerged as a promising target for early diagnosis and cardiovascular risk prediction and is also being considered as a treatment target. Lox-1 was first described as a 50 kDa transmembrane protein in endothelial cells responsible for oxLDL (oxidized LDL) recognition, triggering downstream pathways that intensify atherosclerosis via endothelial dysfunction, oxLDL uptake, and apoptosis. Lox-1 is also expressed in platelets, where it enhances platelet activation, adhesion to endothelial cells, and ADP-mediated aggregation, thereby favoring thrombus formation. Lox-1 was also identified in cardiomyocytes, where it was implicated in the development of cardiac fibrosis and myocyte apoptosis, the main determinants of cardiac recovery following an ischemic insult. Together, these findings have revealed that Lox-1 is implicated in all the main steps of atherosclerosis and has encouraged the development of immunoassays for measurement of sLox-1 (serum levels of soluble Lox-1) to be used as a potential CVD biomarker. Finally, the recent development of synthetic Lox-1 inhibitors and neutralizing antibodies with promising results in animal models has made Lox-1 a target for drug development. In this review, we discuss the main findings regarding the role of Lox-1 in the development, diagnosis, and therapeutic strategies for CVD prevention and treatment.

Cell therapy is an exciting approach to repairing the injured heart. Adult cardiac progenitor cells (aCPCs) tested in clinical studies, although safe, have demonstrated limited benefit when administered post myocardial infarction (MI). By contrast, our recent preclinical work has shown that intramyocardial injection of human neonatal CPCs (nCPCs) is superior to aCPCs in improving cardiac function following MI. However, the therapeutic effects of nCPCs on circulating heart failure biomarker (HFB) and the relationship between biomarker and cardiac hypertrophy, and cardiac function post MI have not been well documented. In the present study, we tested the hypothesis that there would be some associations between circulating HFB and cardiac hypertrophy, or cardiac dysfunction post MI, treatment with nCPCs would alter these associations. Male nude rats (10-week-old) were subjected to MI induced by ligation of left anterior descending coronary artery followed by intramyocardial injection with vehicle or 106 nCPCs into the ischemic region (n = 11/group). Blood samples were collected pre-MI, and weekly post MI up to 4 weeks to determine the circulating levels of pro-ANP and TNF-a. Cardiac function was measured by using a Millar Mikro-Tip pressure catheter four weeks post MI. Masson's Trichrome staining and image analysis by ImageJ were used to quantitate scar size post-MI. A highly significant correlation between pro-ANP and heart weight was observed in vehicle-treated MI rats (R2 = 0.85, p < .0001); treatment with nCPCs markedly shifted the correlation between proANP and heart weight (R2 = 0.0001, p = .98). Similarly, a significant correlation between pro-ANP and cardiac contractile function was also observed in vehicle-treated rats. Interestingly, nCPC treatment altered the association to weaker and less significant. In addition, treatment with nCPCs reduced the circulating levels of TNF-a and scar size. In summary, circulating pro-ANP could be potentially used to predict functional outcomes for the cell therapy in rodent MI model.

The current coronavirus disease 2019 (COVID-19) pandemic presents a global challenge for managing acutely ill patients and complications from viral infection. Systemic inflammation accompanied by a "cytokine storm," hemostasis alterations and severe vasculitis have all been reported to occur with COVID-19, and emerging evidence suggests that dysregulation of lipid transport may contribute to some of these complications. Here, we aim to summarize the current understanding of the potential mechanisms related to COVID-19 dyslipidemia and propose possible adjunctive type therapeutic approaches that modulate lipids and lipoproteins. Specifically, we hypothesize that changes in the quantity and composition of high-density lipoprotein (HDL) that occurs with COVID-19 can significantly decrease the anti-inflammatory and anti-oxidative functions of HDL and could contribute to pulmonary inflammation. Furthermore, we propose that lipoproteins with oxidized phospholipids and fatty acids could lead to virus-associated organ damage via overactivation of innate immune scavenger receptors. Restoring lipoprotein function with ApoA-I raising agents or blocking relevant scavenger receptors with neutralizing antibodies could, therefore, be of value in the treatment of COVID-19. Finally, we discuss the role of omega-3 fatty acids transported by lipoproteins in generating specialized proresolving mediators and how together with anti-inflammatory drugs, they could decrease inflammation and thrombotic complications associated with COVID-19.

Purpose of reviewTo review recent lecithin:cholesterol acyltransferas (LCAT)-based therapeutic approaches for atherosclerosis, acute coronary syndrome, and LCAT deficiency disorders.Recent findingsA wide variety of approaches to using LCAT as a novel therapeutic target have been proposed. Enzyme replacement therapy with recombinant human LCAT is the most clinically advanced therapy for atherosclerosis and familial LCAT deficiency (FLD), with Phase I and Phase 2A clinical trials recently completed. Liver-directed LCAT gene therapy and engineered cell therapies are also another promising approach. Peptide and small molecule activators have shown efficacy in early-stage preclinical studies. Finally, lifestyle modifications, such as fat-restricted diets, cessation of cigarette smoking, and a diet rich in antioxidants may potentially suppress lipoprotein abnormalities in FLD patients and help preserve LCAT activity and renal function but have not been adequately tested.SummaryPreclinical and early-stage clinical trials demonstrate the promise of novel LCAT therapies as HDL-raising agents that may be used to treat not only FLD but potentially also atherosclerosis and other disorders with low or dysfunctional HDL.

Traditional cardiovascular disease (CVD) risk factors, such as hypertension, dyslipidemia and diabetes do not explain the increased CVD burden in systemic lupus erythematosus (SLE). The oxidized-LDL receptor, LOX-1, is an inflammation-induced receptor implicated in atherosclerotic plaque formation in acute coronary syndrome, and here we evaluated its role in SLE-associated CVD. SLE patients have increased sLOX-1 levels which were associated with elevated proinflammatory HDL, oxLDL and hsCRP. Interestingly, increased sLOX-1 levels were associated with patients with early disease onset, low disease activity, increased IL-8, and normal complement and hematological measures. LOX-1 was increased on patient-derived monocytes and low-density granulocytes, and activation with oxLDL and immune-complexes increased membrane LOX-1, TACE activity, sLOX-1 release, proinflammatory cytokine production by monocytes, and triggered the formation of neutrophil extracellular traps which can promote vascular injury. In conclusion, perturbations in the lipid content in SLE patients' blood activate LOX-1 and promote inflammatory responses. Increased sLOX-1 levels may be an indicator of high CVD risk, and blockade of LOX-1 may provide a therapeutic opportunity for ameliorating atherosclerosis in SLE patients.

LCAT converts free cholesterol to cholesteryl esters in the process of reverse cholesterol transport. Familial LCAT deficiency (FLD) is a genetic disease that was first described by Kaare R. Norum and Egil Gjone in 1967. This report is a summary from a 2017 symposium where Dr. Norum recounted the history of FLD and leading experts on LCAT shared their results. The Tesmer laboratory shared structural findings on LCAT and the close homolog, lysosomal phospholipase A2. Results from studies of FLD patients in Finland, Brazil, Norway, and Italy were presented, as well as the status of a patient registry. Drs. Kuivenhoven and Calabresi presented data from carriers of genetic mutations suggesting that FLD does not necessarily accelerate atherosclerosis. Dr. Ng shared that LCAT-null mice were protected from diet-induced obesity, insulin resistance, and nonalcoholic fatty liver disease. Dr. Zhou presented multiple innovations for increasing LCAT activity for therapeutic purposes, whereas Dr. Remaley showed results from treatment of an FLD patient with recombinant human LCAT (rhLCAT). Dr. Karathanasis showed that rhLCAT infusion in mice stimulates cholesterol efflux and suggested that it could also enhance cholesterol efflux from macrophages. While the role of LCAT in atherosclerosis remains elusive, the consensus is that a continued study of both the enzyme and disease will lead toward better treatments for patients with heart disease and FLD.

Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD.

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.