lipoprotein-X Research Articles

A High-Throughput NMR Method for Lipoprotein-X Quantification.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

FRI100 Lipoprotein X In Graft Vs. Host Disease Affecting Liver

Abstract Disclosure: D. Vedamurthy: None. C. Vitali: None. M. Cuchel: None. D. Soffer: None. D. Rader: None. Introduction: Lipoprotein-X (LpX) is an abnormal lipoprotein rich in phospholipid and unesterified cholesterol (UC) found in patients with cholestatic liver disease, lecithin-cholesterol acyl transferase (LCAT) deficiency, and after lipid emulsion infusion. LpX has been detected in patients with graft-versus-host disease (GVHD) of the liver. Cholestasis leads to the accumulation of bile salts and UC in the circulation, promoting the formation of LpX. This condition is often not well-recognized, partly because the detection of LpX is technically challenging and requires specialized laboratory assays. Case Description: A 66-year lady with acute myeloblastic leukemia (AML) underwent an allogeneic matched unrelated donor stem cell transplant (SCT) which was complicated by graft-versus-host disease (GVHD) affecting the liver, eyes, lungs, and skin. Past medical history includes hypertension. Family history was significant for coronary artery disease in the mother. She is a reformed smoker. Her post-transplant course was highly complicated with infections, GVHD affecting the liver, eyes, lungs, and skin, and vascular events including myocardial infarction and stroke. Laboratory testing revealed abnormal liver enzymes (ALT=268, AST=173, ALKP =402 U/L), and a markedly abnormal lipid profile (TC=500, Non-HDL-C=358, TG= 345 mg/dL). Her thyroid panel and viscosity were normal, but apolipoprotein B levels were discordantly low (77mg/dL). The ratio of ApoB: Non-HDL cholesterol was abnormally low (0.215). She was started on ezetimibe, evolocumab, and omega-3 fatty acids by her neurovascular team for cardiovascular risk reduction. A physical exam revealed a thin, frail woman with mild oral and nail GVHD changes. The rest of the exam was notable for the abdomen showing chronic patchy hyperpigmentation with sclerodermatous changes anteriorly and posteriorly. She had no significant focal neurologic deficits. Her lipid levels were improved with therapy, but TC, non-HDL-C, UC, and triglycerides levels were still elevated (TC=353, non-HDL=258, UC=163, TG=414 mg/dL), UC: TC ratio was high (0.46). FPLC separation of TRL-depleted plasma revealed the presence of LpX. LpX is not very atherogenic, and we recommended continuing current medical therapy as the serum viscosity was within normal limits. Conclusion: The presence of LpX should be suspected in the setting of GVHD accompanied by markedly elevated TC. LpX may cause hyperviscosity, which can induce end-organ ischemia. While the direct assessment of LpX is challenging and requires specialized techniques, ApoB and non-HDL-C levels are available in most clinical laboratories, and a very low ApoB: Non-HDL-C ratio in GVHD patients may indicate the presence of LpX. The treatment goal is to resolve the underlying causes of cholestasis. If hyperviscosity is present, Lp-X can be acutely treated by plasma exchange or lipoprotein apheresis. Presentation: Friday, June 16, 2023

The necessity for improving lipid testing reagents: A real world study

BackgroundWe investigated the interference of vitamin C (VitC), glycerol fructose, lipoprotein X (LpX) and lipemia on the analysis of serum lipids. MethodsSerum were collected from 44 patients with VitC infusion, serum lipid concentrations before and after VitC auto-oxidation were compared. Serum of 31 patients with glycerol fructose infusion were collected, triglycerides (TG) measured by glycerol blanking and non-blanking reagents were compared. Forty-four serum samples suspected to contain LpX were collected, LDL-C measured by reagents from five manufacturers were compared. Lipemia samples were collected, LDL-C measured using five different reagents were compared. The interference rate was considered unacceptable if it was greater than 1/2 total allowable error (TEa). ResultsIn patients with VitC infusion, the interference rates of TG and total cholesterol (TC) were −59% (−123%, −28%) and −15% (−21%, −11%), respectively. In patients with glycerol fructose infusion, the interference rate of TG was 13% (4%, 113%). LpX interference led to increased LDL-C results for most reagents. Lipemia caused great interference with LDL-C analysis. ConclusionVitC, glycerol fructose, LpX and lipemia significantly interfered with lipid assays. The reagent formulation should be improved to get reliable results.

Abnormal Lipoproteins Trigger Oxidative Stress-Mediated Apoptosis of Renal Cells in LCAT Deficiency.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease caused by the loss of function mutations in the LCAT gene. LCAT deficiency is characterized by an abnormal lipoprotein profile with severe reduction in plasma levels of high-density lipoprotein (HDL) cholesterol and the accumulation of lipoprotein X (LpX). Renal failure is the major cause of morbidity and mortality in FLD patients; the pathogenesis of renal disease is only partly understood, but abnormalities in the lipoprotein profile could play a role in disease onset and progression. Serum and lipoprotein fractions from LCAT deficient carriers and controls were tested for renal toxicity on podocytes and tubular cells, and the underlying mechanisms were investigated at the cellular level. Both LpX and HDL from LCAT-deficient carriers triggered oxidative stress in renal cells, which culminated in cell apoptosis. These effects are partly explained by lipoprotein enrichment in unesterified cholesterol and ceramides, especially in the HDL fraction. Thus, alterations in lipoprotein composition could explain some of the nephrotoxic effects of LCAT deficient lipoproteins on podocytes and tubular cells.

Dyslipidemia and its features in primary biliary cholangitis

Primary biliary cholangitis, previously known as primary biliary cirrhosis, is a rare autoimmune liver disease that mostly affects women. Disorder of biliary excretion and enterohepatic circulation of bile acids in patients with primary biliary cholangitis in the early stages of the disease leads to their insufficient flow into the intestine and ingestion of bile acids into the general bloodstream. Insufficient intake of bile acids into the duodenum contributes to malabsorption, energetic malnutrition, and a slowly progressing weight loss. Pathophysiological mechanisms of the development of weight loss are related to impaired emulsification of fats and decreased absorption of hydrolyzed products - fatty acids and monoglycerides, steatorrhea in patients with primary biliary cholangitis as well as to dysbiosis of intestinal microflora. Already in the early stages of the disease, this leads to accelerated ß-oxidation of fatty acids, which is aimed at compensating for the developing nutritional energy deficiency. Entry of bile acids into the general bloodstream in primary biliary cholangitis is accompanied by dyslipidemia. The mechanism of hyperlipidemia in patients with primary biliary cholangitis differs from that in other conditions because along with the increase in total cholesterol, there is an increase in high-density lipoproteins and the appearance of unusual lipoprotein X (Lp-X). The appearance of the latter is most likely a protective reaction of the body aimed at the inactivation of bile acids detergent action on membrane structures of blood-forming elements and vascular endotheliocytes. Exactly bile acids, but not the content of total cholesterol, correlates with the level of lipoprotein X and determine its formation. Concomitant hypercholesterolemia in patients with primary biliary cholangitis is also aimed at neutralizing the detergent action of bile acids that entered the general bloodstream and is most likely a compensatory reaction of the body. “Abnormal” hypercholesterolemia in primary biliary cholangitis can serve as a model system to search for and develop new ways of dyslipidemia treatment, as it proceeds without increasing the frequency of cardiovascular events.

Features of Lipid Metabolism Disorders in Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC), previously known as primary biliary cirrhosis, is an autoimmune liver disease that mostly affects women. A progressive disorder in the processes of bile secretion and enterohepatic bile salts circulation in patients with PBC already in its early stages, leading to an insufficient release of bile acids into the bowel and their entry into the systemic circulation. Insufficient bile acids released into the duodenum contributes to the development of malabsorption, energy malnutrition, and slowly progressive weight loss. The pathophysiological mechanisms of weight loss and its slow progression are associated with the deterioration of the fat emulsification processes and with the reduced absorption of hydrolyzed products, such as fatty acids and monoglycerides, with steatorrhea in patients with PBC, as well as in those with gut dysbiosis. Just in the early stages of the disease, this results in accelerated fatty acid β-oxidation that is aimed at compensating for progressive energy malnutrition. The entry of bile acids into the systemic circulation in PBC is accompanied by dyslipidemia. The mechanism of hyperlipidemia in patients with PBC differs from that in other conditions because along with an increase in total cholesterol (TC), there are elevated high-density lipoprotein levels and the appearance of unusual lipoprotein X (Lp-X). The appearance of Lp-X is most likely to be the body's protective reaction to inactivate the detergent effect of bile acids on the membrane structures of blood corpuscles and vascular endothelial cells. It is bile acids, rather than TC levels, that correlate with the content of Lp-X and determine its formation. Concomitant hypercholesterolemia in patients with PBC is also aimed at neutralizing the detergent effect of bile acids that have entered the systemic circulation and is most likely a compensatory reaction of the body. "Anomalous" hypercholesterolemia in PBC can serve as a model system for the search and development of new methods for the treatment of dyslipidemia since it occurs without an increase in the incidence of cardiovascular events.

More Than Just Heart Disease: Predicting the Presence of Lipoprotein X using Lipid Panel Results

Abstract Cholestatic liver diseases, such as primary sclerosing cholangitis and primary biliary cirrhosis, can lead to serum accumulation of lipoprotein X (LpX). LpX is a multilamellar particle high in cholesterol but lacking structural apolipoproteins A1 or B. The absence of ApoB results in no negative feedback on cholesterol biosynthesis and prevents LpX clearance from the liver. While clinical signs and symptoms typically precede laboratory findings, it is possible that in medically complex patients the identification of LpX could be the first observation of cholestatic liver disease. Traditional laboratory methods are insufficient to identify LpX as it is of similar density to low-density lipoprotein (LDL). LpX contains a high concentration of cholesterol which is erroneously reported as LDL-C by routine clinical methods. As LpX is a rare complication of liver disease, clinicians may presume the elevation is a coincidental familial hypercholesterolemia rather than a sequela of liver disease. Currently, lipoprotein gel electrophoresis is the only laboratory method to identify LpX. In this method only performed in specialty lipid laboratories, LpX is readily identified by its unique reverse electrophoretic mobility relative to other lipoproteins. The objective of this study was to characterize lipid panels from LpX-positive samples and develop a suitable mechanism to identify LpX-containing samples with good clinical validity. From 21,377 clinical electrophoresis results reported between Nov 2011 to Nov 2021, LpX was identified in 157 serum samples. Overall, patients with LpX were younger (median 44y vs. 55y, p<0.0001) with significantly higher total cholesterol (812mg/dL vs 190mg/dL, p<0.0001) and lower high density lipoprotein-cholesterol (HDL-C; 3mg/dL vs 45mg/dL, p<0.0001). Data were randomly split (70/30) into training (n=14,964) and testing (n=6,413) cohorts. Receiver operator characteristic curve analysis identified optimal thresholds of 22.5 mg/dL HDL-C (AUC = 0.94) and 378.5 mg/dL total cholesterol (AUC = 0.91), as well as a nonHDL-C/HDL-C ratio of 9.2 (AUC = 0.995). Applying these cutoffs to the testing cohort achieved a sensitivity/specificity of 98%/81% for HDL-C, 96%/87% for total cholesterol, and 98%/94% for nonHDL-C/HDL-C ratio. A multivariate model combining these three parameters showed a sensitivity/specificity of 97%/85%, respectively. In conclusion, low HDL-C, elevated total cholesterol and a ratio of nonHDL-C/HDL-C >9.2 are associated with the presence of LpX. The ratio of nonHDL-C/HDL-C is the most sensitive and specific predictor of LpX. If confirmed in other cohorts, laboratories could include a reporting comment on lipid panels with a nonHDL-C/HDL-C ratio >9.2 cautioning a high suspicion for the presence of LpX and recommending confirmatory testing. It may also be prudent to caution that LDL-C results may not be accurate. In conclusion, identifying patients with high suspicion of LpX based on abnormal lipid panel results may aide in clinical diagnosis, even when an assay to detect LpX is not readily available.

A Simple Fluorescent Cholesterol Labeling Method to Cryoprotect and Detect Plasma Lipoprotein-X.

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.

*Lipoprotein X hyperlipidemia and Its Complications in a Patient with Primary Sclerosing Cholangitis

Lead Author's Financial Disclosures Nothing to disclose. Study Funding None. Background/Synopsis We reviewed a case of a young man referred for management of severe hypercholesterolemia and bilateral palpebral xanthelasma. This is intended to highlight key features in diagnosis and management of severe hypercholesterolemia due to LpX from biliary obstructive disease. Objective/Purpose To review clinical management of severe hypercholesterolemia due to LpX. Methods Clinical management at tertiary care lipid program. Results A 22-year-old slim male with history of Wilson's disease, Crohn's disease, and primary sclerosing cholangitis (PSC) was referred for management of severe hypercholesterolemia and progressive symmetric palpebral xanthelasmas. There is no family history of severe hypercholesterolemia or premature atherosclerotic cardiovascular disease (ASCVD), nor were any other secondary causes identified. He was successfully treated with cholestyramine for pruritis which resulted in marked total cholesterol reduction and increased triglyceride (TG) level. Conclusions Very severe hypercholesterolemia with total cholesterol levels >500 mg/dL is a rare condition. Levels this high are typically associated with homozygous familial hypercholesterolemia (FH) due to an increased level of atherogenic LDL cholesterol (LDL-C). However, other causes need to be considered in the right clinical context, such as lipoprotein fraction lipoprotein X (LpX) in an individual with obstructive biliary disease. LpX is a consequence of ``spillover'' of hepatic free cholesterol (FC)/phospholipid complexes in disc- shaped lipoproteins which do not contain apoB. Consequently, there is wide discordance between apoB and the calculated LDL-C and high serum FC levels. The diagnosis is typically made in the right clinical context without FC measurement since this test is not commercially available. Hypercholesterolemia due to LpX is not associated with increased ASCVD risk, but its presence can be a marker of the severity of liver disease and cause other medical complications including hyperviscosity syndrome, pulmonary embolism and cholesteroloma.Our patient's marked increase in total cholesterol without severe increase in apoB suggested progression of PSC and prompted referral back to his primary Hepatology team. Additional cholesterol-lowering pharmacotherapy is not indicated for hypercholesterolemia due to LpX without hyperviscosity. Nothing to disclose.

The HDL mimetic CER-001 remodels plasma lipoproteins and reduces kidney lipid deposits in inherited lecithin:cholesterol acyltransferase deficiency.

BackgroundKidney failure is the major cause of morbidity and mortality in familial lecithin:cholesterol acyltransferase deficiency (FLD), a rare inherited lipid disorder with no cure. Lipoprotein X (LpX), an abnormal lipoprotein, is primarily accountable for nephrotoxicity.MethodsCER‐001 was tested in an FLD patient with dramatic kidney disease for 12 weeks.ResultsInfusions of CER‐001 normalized the lipoprotein profile, with a disappearance of the abnormal LpX in favour of normal‐sized LDL. The worsening of kidney function was slowed by the treatment, and kidney biopsy showed a slight reduction of lipid deposits and a stabilization of the disease. In vitro experiments demonstrate that CER‐001 progressively reverts lipid accumulation in podocytes by a dual effect: remodelling plasma lipoproteins and removing LpX‐induced lipid deposit.ConclusionThis study demonstrates that CER‐001 may represent a therapeutic option in FLD patients. It also has the potential to be beneficial in other renal diseases characterized by kidney lipid deposits.

Detection of Lipoprotein X (LpX): A challenge in patients with severe hypercholesterolaemia

Detection of Lipoprotein X (LpX): A challenge in patients with severe hypercholesterolaemia

Clinical laboratory characteristics of patients with obstructive jaundice accompanied by dyslipidemia

BackgroundAbnormal lipid metabolism manifests as hypercholesterolemia in patients with obstructive jaundice due to lipoprotein X (LpX). Our aim was to explore the clinical laboratory characteristics of patients with obstructive jaundice accompanied by dyslipidemia in a large number of samples. MethodsA total of 665 patients with obstructive jaundice were included and categorized into two groups (with/without dyslipidemia) based on the ratio of the sum of HDL-c and LDL-c to total cholesterol [(HDL-c + LDL-c)/TC] with a cut-off value of 0.695. Laboratory liver, kidney, and blood lipid parameters were determined. Cholesterol composition assessment was performed by ultracentrifugation and high-performance liquid chromatography (UC-HPLC), and serum protein profiles were analyzed by capillary electrophoresis. ResultsLiver function in patients with obstructive jaundice accompanied by dyslipidemia was more aggravated than that in patients with simple obstructive jaundice (P < 0.05). The (HDL-c + LDL-c)/TC ratio was negatively correlated with bilirubin levels (P < 0.05). In addition, the difference in ApoB/LDL-c ratios was statistically significant between the obstructive jaundice accompanied by dyslipidemia group and healthy control group (P < 0.05). The LDL-c concentration determined by the UC-HPLC method was more than five times that determined by the enzymatic method (P < 0.05). Bisalbuminemia was found in 43 of 60 patients with obstructive jaundice accompanied by hypercholesterolemia. ConclusionsIn patients with obstructive jaundice, the decreased (HDL-c + LDL-c)/TC ratio may be a novel marker to identify dyslipidemia secondary to LpX. The decreased ratio was associated with poor liver function and indicated disease progression.

(PSEUDO)HYPONATREMIA SECONDARY TO LIPOPROTEIN X

SESSION TITLE: Medical Student/Resident Critical Care Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Hyponatremia can be associated with clinically significant disease and often portends worse outcomes, significant hospital morbidity, increased length of stay and hospital cost.[1] Pseudohyponatremia, may be a culprit in overusing hospital resources and unnecessary workup. Here we present a case of altered mental status (AMS) thought initially to be due to hyponatremia. CASE PRESENTATION: A 58 year-old Caucasian male with a history of glioblastoma multiforme (GBM) treated with resection, radiation and temozolomide, on chronic tube feeds (TF) through gastrostomy tube presented with two weeks of AMS. The spouse reported maintenance of TF but was unsure of how much free water she was actually giving. The patient’s exam was significant for blood pressure of 89/60 mmHg, tachycardia at 101 beats per minute, he had lethargy with response to painful stimuli, significant scleral icterus, dry mucous membranes with no gross motor deficits and unremarkable lung and cardiac exams. His workup was significant for sodium of 120 mmol/L, blood urea nitrogen of 48 mg/dL, creatinine of 1.5 mg/dL, direct hyperbilirubinemia of 47 mg/dL (thought to be due to hepatic injury from temozolomide), aspartate aminotransferase of 1,025 U/L, alanine aminotransferase of 595 U/L, alkaline phosphatase of >1,200 U/L, undetectable ammonia level and a negative drug screen. He was started on intravenous fluids and lactulose without any clinical improvement and minimal change in sodium levels. Further workup showed a serum osmolality of 315 mOs/kg (which pointed towards a pseudohyponatremia picture), triglycerides of 343 mg/dL, cholesterol of 1,652 mg/dL, LDL of 24 mg/dL. Full lipoprotein profile and lipid electrophoresis identified lipoprotein X (LpX) which accounted for the observed pseudohyponatremia. MRI head showed possible recurrent GBM without intracranial shift, thought to be the culprit behind his AMS. DISCUSSION: LpX appears in the serum secondary to cholestasis. It is a result of reflux of bile acid into the serum that combines with albumin to form LpX. It is also composed of small amounts of triglycerides, free cholesterol and apolipoprotein C.[2] Interestingly, LpX was not found to be associated with ischemic heart disease. This is thought to be due to a particle size of 18-25 nm, which prevents it from incorporating into the endothelium. It may even be protective against LDL induced atherosclerosis as it decreases LDL oxidation.[3] Thus, lipid lowering medication may not be needed. It is important to note that LpX may cause a hyperviscosity syndrome and could require lipopheresis. CONCLUSIONS: Our case highlights LpX as a rare cause of pseudohyponatremia and the importance of differentiating between true hyponatremia and pseudohyponatremia to avoid unnecessary workup and therapy that may increase morbidity and length of hospital stay. Physicians should be aware of this entity and include it in future practice. Reference #1: Corona G, Giuliani C, Parenti G, et al. The Economic Burden of Hyponatremia: Systematic Review and Meta-Analysis. Am J Med. 2016;129(8):823-835.e4. doi:10.1016/j.amjmed.2016.03.007 Reference #2: Fellin R, Manzato E. Lipoprotein-X fifty years after its original discovery. Nutr Metab Cardiovasc Dis. 2019;29(1):4-8. doi:10.1016/j.numecd.2018.09.006 Reference #3: Chang PY, Lu SC, Su TC, et al. Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation. J Lipid Res. 2004;45(11):2116-2122. doi:10.1194/jlr.M400229-JLR200 DISCLOSURES: No relevant relationships by Youssef Abouleish, source=Web Response Speaker/Speaker's Bureau relationship with Genentech Please note: $5001 - $20000 Added 04/01/2020 by Joshua Sill, source=Web Response, value=Honoraria

Characterization of LP-Z Lipoprotein Particles and Quantification in Subjects with Liver Disease Using a Newly Developed NMR-Based Assay.

Background: Lipoprotein particles with abnormal compositions, such as lipoprotein X (LP-X) and lipoprotein Z (LP-Z), have been described in cases of obstructive jaundice and cholestasis. The study objectives were to: (1) develop an NMR-based assay for quantification of plasma/serum LP-Z particles, (2) evaluate the assay performance, (3) isolate LP-Z particles and characterize them by lipidomic and proteomic analysis, and (4) quantify LP-Z in subjects with various liver diseases. Methods: Assay performance was assessed for linearity, sensitivity, and precision. Mass spectroscopy was used to characterize the protein and lipid content of isolated LP-Z particles. Results: The assay showed good linearity and precision (2.5–6.3%). Lipid analyses revealed that LP-Z particles exhibit lower cholesteryl esters and higher free cholesterol, bile acids, acylcarnitines, diacylglycerides, dihexosylceramides, lysophosphatidylcholines, phosphatidylcholines, triacylglycerides, and fatty acids than low-density lipoprotein (LDL) particles. A proteomic analysis revealed that LP-Z have one copy of apolipoprotein B per particle such as LDL, but less apolipoprotein (apo)A-I, apoC3, apoA-IV and apoC2 and more complement C3. LP-Z were not detected in healthy volunteers or subjects with primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, or type 2 diabetes. LP-Z were detected in some, but not all, subjects with hypertriglyceridemia, and were high in some subjects with alcoholic liver disease. Conclusions: LP-Z differ significantly in their lipid and protein content from LDL. Further studies are needed to fully understand the pathophysiological reason for the enhanced presence of LP-Z particles in patients with cholestasis and alcoholic liver disease.

Monocyte phenotyping and management of lipoprotein X syndrome

Accumulation of lipoprotein X (LpX) in blood can cause severe hypercholesterolemia and cutaneous xanthomas. Monocytes sensitively sense lipid changes in circulation and contribute to inflammation. However, how monocytes respond to LpX is undefined. We examined the phenotype of monocytes from a patient, who had LpX, severe hypercholesterolemia, and extensive cutaneous xanthomas, and effects of semiselective plasmapheresis therapy (SPPT). Fluorescence-activated cell sorting and adhesion assays were used to examine monocyte phenotype and exvivo oxidized low-density lipoprotein uptake and adhesion in the patient before and after treatment with SPPT. Effects of plasma from the patient on the phenotype and adhesion of monocytes from a healthy participant were determined. SPPT improved hypercholesterolemia and cutaneous xanthomas. Before treatment, the patient had lower frequency of nonclassical monocytes but higher frequency of intermediate monocytes than the control participant. Before treatment, monocytes from the patient with LpX showed more intracellular lipid accumulation, alterations in several cell surface markers and intracellular cytokines, as well as enhanced oxidized low-density lipoprotein uptake and reduced adhesion compared with control. After SPPT, the phenotypes of monocytes from the patient with LpX were similar to control monocytes. Incubation with plasma from the patient before treatment as compared with plasma from the control participant or the patient after treatment increased CD11c expression and adhesion of monocytes from a healthy participant. LpX-induced hypercholesterolemia increased lipid accumulation and altered the phenotype of monocytes, which may contribute to cutaneous xanthoma development. Removal of LpX by SPPT reduced lipid accumulation and improved monocyte phenotype, likely contributing to xanthoma resolution.

SAT-572 Extremely Elevated Plasma Lipoprotein X Level Secondary to Alcoholic Cholestasis

BackgroundMarked elevations of plasma lipoprotein X (Lp-X) levels have been reported in patients with cholestasis due to primary biliary cirrhosis, pancreatic cancer, hepatitis C, and quetiapine. We now report a patient with extreme elevation of plasma Lp-X due to alcohol-induced cholestasis.Case PresentationA 44 year-old African American male presented with painless jaundice and fatigue for one week. He denied nausea, vomiting, diarrhea, change in stool or urine color, or weight loss. He consumes 720-1080 mL of beer (2-3 cans) every night and admitted to heavier alcohol consumption in the past. On physical examination he had scleral icterus and hepatomegaly but no xanthomas or xanthelasmas.His serum total cholesterol was 1,126 mg/dL (normal range, 120-199 mg/dL), triglycerides were 238 mg/dL (50-150 mg/dL), calculated LDL-cholesterol was 1,072 mg/dL (<100 mg/dL), and HDL-cholesterol was 6 mg/dL (>39 mg/dL). His serum AST, 162 IU/L (10-50 IU/L); ALT, 79 IU/L (10-50 IU/L); alkaline phosphatase, 1,058 IU/L (40-129 IU/L); total bilirubin, 18.8 mg/dL (0.2-1.3 mg/dL); direct bilirubin, 13.5 mg/dL (0-0.3 mg/dL); and gamma glutamyl transferase, 4,583 IU/L (8-61 IU/L) were markedly elevated. His blood alcohol level was 34 mg/dL (not detected), sodium 124 mmol/L (135-145 mmol/L), and platelet count was 84,000/µL (150,000-459,000/µL). His TSH 2.89 µIU/mL (0.4-4.5 µIU/mL), UA without proteinuria, HBV immunized, HCV negative, and anti-mitochondrial antibody negative. CT abdomen revealed hepatic steatosis and gallbladder swelling without evidence of obstruction. MRCP showed cirrhosis without primary sclerosing cholangitis. Serum lipoprotein electrophoresis confirmed the presence of Lp-X. On day 3 of hospitalization, his cholestasis improved and his serum total bilirubin 10.0 mg/dL, direct bilirubin 7.4 mg/dL, AST 108 IU/L, ALT 66 IU/L, and alkaline phosphatase 663 IU/L had improved. The patient was advised to abstain from all alcohol consumption. Telephone follow up 2 months later with his wife revealed that he had stopped drinking alcohol and that his jaundice had resolved.ConclusionsAlthough alcohol-induced cholestasis is a well-recognized entity, such presentation with extreme elevations of Lp-X has not been previously reported. In such patients, it is important to establish whether extreme hypercholesterolemia is due to LDL or Lp-X since, as opposed to LDL, Lp-X elevations are not considered to be atherogenic.

LCAT protects against Lipoprotein-X formation in a murine model of drug-induced intrahepatic cholestasis.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL‐C levels, low plasma cholesterol esterification, and the formation of Lipoprotein‐X (Lp‐X), an abnormal cholesterol‐rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp‐X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp‐X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp‐X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT‐deficient (KO), wild type (WT), and LCAT‐transgenic (Tg) mice by gavaging mice with alpha‐naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT‐Tg mice, however, prevented the formation of Lp‐X and other plasma lipid abnormalities in WT and LCAT‐KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp‐X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp‐X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp‐X in other diseases besides FLD.

Lipid Profile Rather Than the LCAT Mutation Explains Renal Disease in Familial LCAT Deficiency

Renal complications are the major cause of morbidity and mortality in patients with familial lecithin–cholesterol acyltransferase (LCAT) deficiency (FLD). We report three FLD patients, two of them siblings—only one of whom developed renal disease—and the third case being a young man with early renal disease. The aim of this study was to analyze the clinical characteristics and possible mechanisms associated with renal disease in these patients. Plasma lipid levels, LCAT activity, lipoprotein particle profile by NMR and FPLC, free and esterified cholesterol, presence of lipoprotein X (LpX) and DNA sequencing in the three FLD patients have been determined. The three cases presented clinical characteristics of FLD, although only one of the siblings developed renal disease, at 45 years of age, while the other patient developed the disease in his youth. Genetic analysis revealed new missense homozygous mutations, p.(Ile202Thr) in both siblings and p.(Arg171Glu) in the other patient. Lipoprotein particle analysis showed that the two patients with renal disease presented higher numbers of small very low-density lipoprotein (VLDL) and a higher concentration of triglycerides in VLDL. This study reports three new cases of LCAT deficiency, not previously described. Renal disease is not only dependent on LCAT deficiency, and could be due to the presence of VLDL particles, which are rich in triglycerides, free cholesterol and LpX.

Detection of Lipoprotein X (LpX): A challenge in patients with severe hypercholesterolaemia.

Lipoprotein X (LpX) is an abnormal lipoprotein fraction, which can be detected in patients with severe hypercholesterolaemia and cholestatic liver disease. LpX is composed largely of phospholipid and free cholesterol, with small amounts of triglyceride, cholesteryl ester and protein. There are no widely available methods for direct measurement of LpX in routine laboratory practice. We present the heterogeneity of clinical and laboratory manifestations of the presence of LpX, a phenomenon which hinders LpX detection. The study was conducted on a 26-year-old female after liver transplantation (LTx) with severely elevated total cholesterol (TC) of 38 mmol/L and increased cholestatic liver enzymes. TC, free cholesterol (FC), cholesteryl esters (CE), triglycerides, phospholipids, HDL-C, LDL-C, and apolipoproteins AI and B were measured. TC/apoB and FC:CE ratios were calculated. Lipoprotein electrophoresis was performed using a commercially available kit and laboratory-prepared agarose gel. Commercially available electrophoresis failed to demonstrate the presence of LpX. Laboratory-prepared gel clearly revealed the presence of lipoproteins with γ mobility, characteristic of LpX. The TC/apoB ratio was elevated and the CE level was reduced, confirming the presence of LpX. Regular lipoprotein apheresis was applied as the method of choice in LpX disease and a bridge to reLTx due to chronic liver insufficiency. The detection of LpX is crucial as it may influence the method of treatment. As routinely available biochemical laboratory tests do not always indicate the presence of LpX, in severe hypercholesterolaemia with cholestasis, any discrepancy between electrophoresis and biochemical tests should raise suspicions of LpX disease.

Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo

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.