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Alan T. Remaley - One of the best experts on this subject based on the ideXlab platform.
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lcat protects against Lipoprotein X formation in a murine model of drug induced intrahepatic cholestasis
Pharmacology Research & Perspectives, 2020Co-Authors: Marcelo Amar, Lita A Freeman, Takafumi Nishida, Maureen Sampson, Milton Pryor, Boris Vaisman, Edward B Neufeld, Sotirios K Karathanasis, Alan T. RemaleyAbstract: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.
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Plasma Lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment eX vivo.
Journal of lipid research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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
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plasma Lipoprotein X quantification on filipin stained gels monitoring recombinant lcat treatment eX vivo
Journal of Lipid Research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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.
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Abstract 16097: The Effects of MEDI6012 on Lipoproteins in Familial LCAT Deficiency Patients and a New NMR Method for Quantifying Lipoprotein-X
Circulation, 2017Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Boris Vaisman, Sotirios K Karathanasis, John Le Lay, Irina Shalaurova, James D. Otvos, Alan T. RemaleyAbstract:Introduction: Lecithin:cholesterol acyltransferase (LCAT), a key enzyme for HDL maturation, catalyzes esterification of cholesterol to cholesteryl ester in plasma. Functional LCAT is absent in patients with Familial LCAT Deficiency (FLD), an autosomal recessive disorder characterized by very low HDL-cholesterol, anemia, nephropathy, and circulating Lipoprotein-X (LP-X). LP-X is an abnormal multilamellar particle, enriched in phospholipids and unesterified cholesterol, and is nephrotoXic in mice. Objectives: To characterize the effects of MEDI6012 (rhLCAT) on LP-X in plasma from FLD patients and to compare LP-X measured by agarose gel electrophoresis with a novel assay utilizing nuclear magnetic resonance (NMR) spectroscopy. Methods: We incubated MEDI6012 with fresh plasma from FLD patients and investigated changes in Lipoproteins either by Sebia gel electrophoresis or by a novel proton NMR assay on the Vantera analyzer, which detects and quantifies LP-X by eXploiting the unique spectral shape and position...
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Effect of Recombinant Human Lecithin Cholesterol Acyltransferase Infusion on Lipoprotein Metabolism in Mice
Journal of Pharmacology and Experimental Therapeutics, 2010Co-Authors: Xavier Rousset, Bruce J. Auerbach, Brian R. Krause, Reyn Homan, Boris L. Vaisman, Robert D Shamburek, John A Stonik, G. Csákó, Alan T. RemaleyAbstract:Lecithin cholesterol acyl transferase (LCAT) deficiency is associated with low high-density Lipoprotein (HDL) and the presence of an abnormal Lipoprotein called Lipoprotein X (Lp-X) that contributes to end-stage renal disease. We eXamined the possibility of using LCAT an as enzyme replacement therapy agent by testing the infusion of human recombinant (r)LCAT into several mouse models of LCAT deficiency. Infusion of plasma from human LCAT transgenic mice into LCAT-knockout (KO) mice rapidly increased HDL-cholesterol (C) and lowered cholesterol in fractions containing very-low-density Lipoprotein (VLDL) and Lp-X. rLCAT was produced in a stably transfected human embryonic kidney 293f cell line and purified to homogeneity, with a specific activity of 1850 nmol/mg/h. Infusion of rLCAT intravenously, subcutaneously, or intramuscularly into human apoA-I transgenic mice showed a nearly identical effect in increasing HDL-C approXimately 2-fold. When rLCAT was intravenously injected into LCAT-KO mice, it showed a similar effect as plasma from human LCAT transgenic mice in correcting the abnormal Lipoprotein profile, but it had a considerably shorter half-life of approXimately 1.23 ± 0.63 versus 8.29 ± 1.82 h for the plasma infusion. rLCAT intravenously injected in LCAT-KO mice crossed with human apoLipoprotein (apo)A-I transgenic mice had a half-life of 7.39 ± 2.1 h and increased HDL-C more than 8-fold. rLCAT treatment of LCAT-KO mice was found to increase cholesterol effluX to HDL isolated from mice when added to cells transfected with either ATP-binding cassette (ABC) transporter A1 or ABCG1. In summary, rLCAT treatment rapidly restored the normal Lipoprotein phenotype in LCAT-KO mice and increased cholesterol effluX, suggesting the possibility of using rLCAT as an enzyme replacement therapy agent for LCAT deficiency.
Lita A Freeman - One of the best experts on this subject based on the ideXlab platform.
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lcat protects against Lipoprotein X formation in a murine model of drug induced intrahepatic cholestasis
Pharmacology Research & Perspectives, 2020Co-Authors: Marcelo Amar, Lita A Freeman, Takafumi Nishida, Maureen Sampson, Milton Pryor, Boris Vaisman, Edward B Neufeld, Sotirios K Karathanasis, Alan T. RemaleyAbstract: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.
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Plasma Lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment eX vivo.
Journal of lipid research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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
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plasma Lipoprotein X quantification on filipin stained gels monitoring recombinant lcat treatment eX vivo
Journal of Lipid Research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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.
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Abstract 16097: The Effects of MEDI6012 on Lipoproteins in Familial LCAT Deficiency Patients and a New NMR Method for Quantifying Lipoprotein-X
Circulation, 2017Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Boris Vaisman, Sotirios K Karathanasis, John Le Lay, Irina Shalaurova, James D. Otvos, Alan T. RemaleyAbstract:Introduction: Lecithin:cholesterol acyltransferase (LCAT), a key enzyme for HDL maturation, catalyzes esterification of cholesterol to cholesteryl ester in plasma. Functional LCAT is absent in patients with Familial LCAT Deficiency (FLD), an autosomal recessive disorder characterized by very low HDL-cholesterol, anemia, nephropathy, and circulating Lipoprotein-X (LP-X). LP-X is an abnormal multilamellar particle, enriched in phospholipids and unesterified cholesterol, and is nephrotoXic in mice. Objectives: To characterize the effects of MEDI6012 (rhLCAT) on LP-X in plasma from FLD patients and to compare LP-X measured by agarose gel electrophoresis with a novel assay utilizing nuclear magnetic resonance (NMR) spectroscopy. Methods: We incubated MEDI6012 with fresh plasma from FLD patients and investigated changes in Lipoproteins either by Sebia gel electrophoresis or by a novel proton NMR assay on the Vantera analyzer, which detects and quantifies LP-X by eXploiting the unique spectral shape and position...
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Abstract 230: Lipoprotein X Causes Renal Disease in LCAT Deficiency
Arteriosclerosis Thrombosis and Vascular Biology, 2016Co-Authors: Edward B Neufeld, Lita A Freeman, Milton Pryor, Boris Vaisman, Alice Ossoli, Seth G. Thacker, Christine A. Brantner, Irina N. Baranova, Nicolás O. Francone, Stephen J. DemoskyAbstract:Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called Lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoXic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, and chemical properties, to wild-type and Lcat -/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of eXogenous LpX was markedly delayed in Lcat -/- mice, which have low HDL but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered eXogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat -/- mice, and induced proteinuria and nephrotoXic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. EXtensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes, where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA 2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal CXl10 eXpression in Lcat -/- mice. In conclusion, LpX is a nephrotoXic particle that in the absence of LCAT induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.
Robert D Shamburek - One of the best experts on this subject based on the ideXlab platform.
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plasma Lipoprotein X quantification on filipin stained gels monitoring recombinant lcat treatment eX vivo
Journal of Lipid Research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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.
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Plasma Lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment eX vivo.
Journal of lipid research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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
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Abstract 16097: The Effects of MEDI6012 on Lipoproteins in Familial LCAT Deficiency Patients and a New NMR Method for Quantifying Lipoprotein-X
Circulation, 2017Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Boris Vaisman, Sotirios K Karathanasis, John Le Lay, Irina Shalaurova, James D. Otvos, Alan T. RemaleyAbstract:Introduction: Lecithin:cholesterol acyltransferase (LCAT), a key enzyme for HDL maturation, catalyzes esterification of cholesterol to cholesteryl ester in plasma. Functional LCAT is absent in patients with Familial LCAT Deficiency (FLD), an autosomal recessive disorder characterized by very low HDL-cholesterol, anemia, nephropathy, and circulating Lipoprotein-X (LP-X). LP-X is an abnormal multilamellar particle, enriched in phospholipids and unesterified cholesterol, and is nephrotoXic in mice. Objectives: To characterize the effects of MEDI6012 (rhLCAT) on LP-X in plasma from FLD patients and to compare LP-X measured by agarose gel electrophoresis with a novel assay utilizing nuclear magnetic resonance (NMR) spectroscopy. Methods: We incubated MEDI6012 with fresh plasma from FLD patients and investigated changes in Lipoproteins either by Sebia gel electrophoresis or by a novel proton NMR assay on the Vantera analyzer, which detects and quantifies LP-X by eXploiting the unique spectral shape and position...
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Effect of Recombinant Human Lecithin Cholesterol Acyltransferase Infusion on Lipoprotein Metabolism in Mice
Journal of Pharmacology and Experimental Therapeutics, 2010Co-Authors: Xavier Rousset, Bruce J. Auerbach, Brian R. Krause, Reyn Homan, Boris L. Vaisman, Robert D Shamburek, John A Stonik, G. Csákó, Alan T. RemaleyAbstract:Lecithin cholesterol acyl transferase (LCAT) deficiency is associated with low high-density Lipoprotein (HDL) and the presence of an abnormal Lipoprotein called Lipoprotein X (Lp-X) that contributes to end-stage renal disease. We eXamined the possibility of using LCAT an as enzyme replacement therapy agent by testing the infusion of human recombinant (r)LCAT into several mouse models of LCAT deficiency. Infusion of plasma from human LCAT transgenic mice into LCAT-knockout (KO) mice rapidly increased HDL-cholesterol (C) and lowered cholesterol in fractions containing very-low-density Lipoprotein (VLDL) and Lp-X. rLCAT was produced in a stably transfected human embryonic kidney 293f cell line and purified to homogeneity, with a specific activity of 1850 nmol/mg/h. Infusion of rLCAT intravenously, subcutaneously, or intramuscularly into human apoA-I transgenic mice showed a nearly identical effect in increasing HDL-C approXimately 2-fold. When rLCAT was intravenously injected into LCAT-KO mice, it showed a similar effect as plasma from human LCAT transgenic mice in correcting the abnormal Lipoprotein profile, but it had a considerably shorter half-life of approXimately 1.23 ± 0.63 versus 8.29 ± 1.82 h for the plasma infusion. rLCAT intravenously injected in LCAT-KO mice crossed with human apoLipoprotein (apo)A-I transgenic mice had a half-life of 7.39 ± 2.1 h and increased HDL-C more than 8-fold. rLCAT treatment of LCAT-KO mice was found to increase cholesterol effluX to HDL isolated from mice when added to cells transfected with either ATP-binding cassette (ABC) transporter A1 or ABCG1. In summary, rLCAT treatment rapidly restored the normal Lipoprotein phenotype in LCAT-KO mice and increased cholesterol effluX, suggesting the possibility of using rLCAT as an enzyme replacement therapy agent for LCAT deficiency.
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Human Lecithin:Cholesterol Acyltransferase Deficiency. In Vivo Kinetics of Low-Density Lipoprotein and Lipoprotein-X
Arteriosclerosis thrombosis and vascular biology, 2006Co-Authors: Masato Nishiwaki, Robert D Shamburek, Alan T. Remaley, Katsunori Ikewaki, Giovanni Bader, Hassan Nazih, Minna Hannuksela, H. Bryan BrewerAbstract:Objectives— Lecithin:cholesterol acyltransferase deficiency (LCAT-def) is characterized by low levels of high-density Lipoprotein (HDL) and low-density Lipoprotein (LDL) and the accumulation of Lipoprotein-X (LpX). Despite the low HDL, atherosclerosis is uncommon in LCAT-def. The decreased LDL would be a possible eXplanation but the underlying mechanism is not clear. In addition, the mechanism(s) for LpX accumulation is not known. The aim of the present study is to elucidate the mechanism(s) responsible for the low LDL and determine the plasma kinetics of LpX in LCAT-def. Methods and Results— We conducted a radiotracer study in LCAT-def (n=2) and normal controls (n=10) and a stable isotope study in one patient and other controls (n=7). LCAT-def LDL was catabolized faster than control LDL in the control subjects as well as in LCAT-def patients. Control LDL was catabolized faster in LCAT-def patients than the controls. The production rate of LDL apoLipoprotein B-100 was normal in LCAT-def. The increased LDL apoB-100 catabolism was confirmed by a stable isotope study. LpX was catabolized more slowly in LCAT-def. Conclusions— The decreased LDL in LCAT-def is attributable to an increased catabolism caused by a rapid catabolism of abnormal LDL and an upregulation of LDL receptor pathway. The decreased catabolism of LpX contributes to its accumulation in LCAT-def.
Edward B Neufeld - One of the best experts on this subject based on the ideXlab platform.
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lcat protects against Lipoprotein X formation in a murine model of drug induced intrahepatic cholestasis
Pharmacology Research & Perspectives, 2020Co-Authors: Marcelo Amar, Lita A Freeman, Takafumi Nishida, Maureen Sampson, Milton Pryor, Boris Vaisman, Edward B Neufeld, Sotirios K Karathanasis, Alan T. RemaleyAbstract: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.
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Plasma Lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment eX vivo.
Journal of lipid research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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
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plasma Lipoprotein X quantification on filipin stained gels monitoring recombinant lcat treatment eX vivo
Journal of Lipid Research, 2019Co-Authors: Lita A Freeman, Robert D Shamburek, Maureen Sampson, Edward B Neufeld, Sotirios K Karathanasis, Masaki Sato, Alan T. RemaleyAbstract: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.
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Abstract 230: Lipoprotein X Causes Renal Disease in LCAT Deficiency
Arteriosclerosis Thrombosis and Vascular Biology, 2016Co-Authors: Edward B Neufeld, Lita A Freeman, Milton Pryor, Boris Vaisman, Alice Ossoli, Seth G. Thacker, Christine A. Brantner, Irina N. Baranova, Nicolás O. Francone, Stephen J. DemoskyAbstract:Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called Lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoXic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, and chemical properties, to wild-type and Lcat -/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of eXogenous LpX was markedly delayed in Lcat -/- mice, which have low HDL but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered eXogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat -/- mice, and induced proteinuria and nephrotoXic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. EXtensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes, where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA 2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal CXl10 eXpression in Lcat -/- mice. In conclusion, LpX is a nephrotoXic particle that in the absence of LCAT induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.
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Lipoprotein X Causes Renal Disease in LCAT Deficiency
PloS one, 2016Co-Authors: Alice Ossoli, Lita A Freeman, Milton Pryor, Boris Vaisman, Edward B Neufeld, Seth G. Thacker, Christine A. Brantner, Irina N. Baranova, Nicolás O. Francone, Stephen J. DemoskyAbstract:Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called Lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoXic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of eXogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered eXogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoXic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. EXtensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal CXl10 eXpression in Lcat-/- mice. In conclusion, LpX is a nephrotoXic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.
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catabolism of Lipoprotein X induced by infusion of 10 fat emulsion
Nutrition, 1997Co-Authors: Mikio Abe, T Tashiro, Hideo Yamamori, Mikihiko Kawano, Kazuya Takagi, Yuichi Morishima, Kohji Shirai, Yasushi Saitou, Nobuyuki NakajimaAbstract:The clinical significance of Lipoprotein-X (Lp-X) induced by intravenous infusion of 10% fat emulsion was assessed, with special reference to atherogenesis, by in vitro eXperiment using purified Lp-X from the sera of patients receiving Intralipid 10%. Lp-X appeared after long-term intravenous infusion of 10% fat emulsion in the patients with intestinal fistula due to the anastomotic leakage. To clarify the role of Lp-X in terms of atherogenicity, the cholesterol metabolism of Lp-X in macrophages as scavenger cells and in hepatocytes as parenchymal cells was studied. When [3H]cholesterol-labeled Lp-X or oXidized low-density Lipoprotein (o-LDL) was incubated with J-774 macrophages, the incorporation of Lp-X into macrophages was negligible compared with o-LDL. When Lp-X or high-density Lipoprotein (HDL) was incubated with J-774 macrophages laden with [3H]cholesterol, the release of cholesterol from macrophages was enhanced by Lp-X as well as HDL. When [3H]cholesterol-labeled Lp-X LDL or HDL was incubated with the human hepatoma cell line of Hep G2 cells, the incorporation of Lp-X into Hep G2 cells was less than that of LDL, but similar to that of HDL. From these findings, it is suggested that the catabolism of Lp-X cholesterol generated with intravenous 10% fat emulsion was mediated by hepatocytes rather than by macrophages, indicating that the hyperlipidemia due to increased Lp-X may not be atherogenic.
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intravenous intralipid 10 vs 20 hyperlipidemia and increase in Lipoprotein X in humans
Nutrition, 1992Co-Authors: T Tashiro, Y Mashima, Hideo Yamamori, M Sanada, Masahiko Nishizawa, K OkuiAbstract:To clarify the mechanisms of hyperlipidemia caused by infusion of Intralipid 10%, we compared Lipoprotein metabolism during intravenous Intralipid 10% and Intralipid 20%, which contains only half the amount of egg yolk lecithin for the same content of triglyceride as Intralipid 10%. Ten patients receiving 20 ml.kg-1.day-1 of Intralipid 10% and 10 receiving 10 ml.kg-1.day-1 of Intralipid 20% were fed eXclusively by total parenteral nutrition (TPN) providing 1.1 g amino acid and 30 kcal.kg-1.day-1 for 4-6 wk. Intravenous Intralipid 10% caused a marked increase in low-density Lipoprotein (LDL), together with increases in phospholipid and cholesterol, especially free cholesterol. The progressive increase in Lipoprotein X was in proportion with that of LDL or total lipid, whereas no increase in lipids, LDL, or Lipoprotein X was observed during intravenous Intralipid 20%. A significant increase in apoLipoproteins CIII and E with Intralipid 10% also caused a rise in Lipoprotein X. With Intralipid 20%, however, the alterations in apoLipoproteins were not observed. Lecithin:cholesterol acyltransferase (LCAT) activity was significantly elevated with Intralipid 10 but not 20%. Disappearance of Lipoprotein X after cessation of Intralipid 10% was relatively rapid, and the half-life was 24-60 h. From these findings, the hyperlipidemia with Intralipid 10% was caused almost eXclusively by the increase in Lipoprotein X. The eXcess lecithin may be responsible for the formation of and increase in Lipoprotein X. Furthermore, it was revealed that Intralipid 20% could be safely used without inducing hyperlipidemia.
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increased Lipoprotein X causes hyperlipidemia during intravenous administration of 10 fat emulsion in man
Journal of Parenteral and Enteral Nutrition, 1991Co-Authors: T Tashiro, Y Mashima, Hideo Yamamori, M Sanada, Masahiko Nishizawa, Kazuo Horibe, K OkuiAbstract:To clarify the mechanisms of hyperlipidemia during intravenous Intralipid 10%, Lipoprotein profiles including Lipoprotein X were studied in 13 patients receiving 2.0 g of fat per kilogram per day by Intralipid 10% over a period of 8 weeks. All patients were fed eXclusively by total parenteral nutrition providing 1.1 g of amino acid and 30 kcal/kg per day. Intravenous administration of Intralipid 10% caused a marked increase of low-density Lipoprotein (LDL), phospholipid, and cholesterol, especially free cholesterol, whereas triglyceride, very-low-density Lipoprotein, and high-density Lipoprotein remained within the normal range. Lipid composition of LDL approXimated that of Lipoprotein X progressively with the intravenous Intralipid 10%. Quantification of Lipoprotein X revealed that its increase was proportionate with that of LDL and total lipid. From these findings, hyperlipidemia during intravenous Intralipid 10% is induced almost eXclusively by the increased Lipoprotein X.