Evaluation of two different homogeneous assays for LDL-cholesterol in lipoprotein-X-positive serum.
(1/23)
BACKGROUND: The purpose of this study was to evaluate the performance of two homogeneous assays for LDL-cholesterol (LDL-C), a polyethylene/cyclodextrin (PC) assay and a detergent (D) assay, which are based on different principles, in cholestatic serum. METHODS: We compared serum LDL-C concentrations determined by the two assays for healthy normolipidemic subjects (n = 42) and cholestatic patients (n = 51). LDL-C concentrations obtained with the homogeneous assays were also compared with those obtained by HPLC for patients' sera. In the interference study, conjugated bile acids were added to normal serum, and their effects on the two assays were examined. The effects of lipoprotein-X (LP-X), intermediate-density lipoprotein (IDL), and apolipoprotein (apo) E-rich HDL on the LDL-C assays were also investigated by adding these lipoproteins to normal serum. RESULTS: The LDL-C concentrations obtained with the D assay were higher than those obtained with the PC assay in the serum with high LP-X. The bias for LDL-C vs LP-X in cholestatic serum correlated with LP-X concentration (r = 0.582; P: <0.0001; n = 51). In the interference study, no effect of bile acids on the LDL-C assays was observed. However, the D assay measured 51.0% of the cholesterol in LP-X, whereas no reactivity was observed for LP-X in the PC assay. In addition, the D assay and the PC assay measured IDL-cholesterol at 31.2% and 52.4%, respectively, and measured apo E-rich HDL-C at 7.6% and 17.8%, respectively. CONCLUSIONS: Although both homogeneous LDL-C assays are suitable for most cases, the present study showed that each homogeneous assay has a different limitation for cholestatic serum with gross alterations in lipoproteins. (+info)
Hyperlipidemia and atherosclerosis associated with liver disease in ferrochelatase-deficient mice.
(2/23)
Erythropoietic protoporphyria (EPP) is an inherited disorder of heme synthesis caused by deficiency of the mitochondrial enzyme ferrochelatase. EPP in humans is associated with liver disease, hypertriglyceridemia, and a low level of high density lipoprotein (HDL) cholesterol. To explore consequences of ferrochelatase deficiency in lipid metabolism, we have analyzed hepatic lipid content and plasma lipoprotein levels in chow-fed BALB/c mice homozygous ( fch/fch) or heterozygous ( fch/1) for a point mutation in the ferrochelatase gene and in wild-type controls (1/1). Livers of fch/fch mice show bile duct proliferation and biliary fibrosis, but bile formation is not impaired. The free cholesterol content of fch/fch livers is significantly increased when compared with fch/1 and 1/1 livers. Plasma cholesterol in fch/fch mice (9.9 +/- 6.4 mM) is elevated when compared with fch/1 and 1/1 mice (2.9 +/- 0.2 and 2.5 +/- 0.3 mM, respectively), because of an increased cholesterol content in the very low density lipoprotein-sized fractions, whereas HDL cholesterol is reduced. The ratio of cholesteryl ester to free cholesterol is 4.3 +/- 0.6, 3.3 +/- 0.3, and 0.3 +/- 0.1 in the plasma of 1/1, fch/1, and fch/fch mice, respectively. The latter is not due to reduced lecithin:cholesterol acyltransferase activity in plasma of fch/fch mice but to the presence of lipoprotein-X (Lp-X), a particle composed of bile-type lipids usually seen only in cholestatic conditions. Expression of mdr2, essential for biliary phospholipid/cholesterol secretion, is increased in fch/fch livers. In spite of this, biliary phospholipid/cholesterol secretion is reduced relative to that of bile salts. It is postulated that an inability of bile salts to stimulate lipid secretion adequately leads to formation of Lp-X in this noncholestatic condition. Distinct atherosclerotic lesions were found in aged fch/fch mice.Thus, ferrochelatase deficiency in mice leads to liver disease associated with altered hepatic lipid metabolism, a characteristic hyperlipidemia, and development of atherosclerosis.-Bloks, V. W., T. Plosch, H. van Goor, H. Roelofsen, J. Baller, R. Havinga, H. J. Verkade, A. van Tol, P. L. M. Jansen, and F. Kuipers. Hyperlipidemia and atherosclerosis associated with liver disease in ferrochelatase-deficient mice. J. Lipid Res. 2001. 42: 41;-50. (+info)
Lipoprotein-X stimulates monocyte chemoattractant protein-1 expression in mesangial cells via nuclear factor-kappa B.
(3/23)
BACKGROUND: Lipoprotein-X (Lp-X) is an abnormal lipoprotein found in the plasma of patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. The majority of patients with this disorder develop progressive glomerulosclerosis. One key event in the pathogenesis of glomerulosclerosis is the infiltration of monocytes into affected glomeruli. Mesangial cells can synthesize and secrete monocyte chemoattractant protein-1 (MCP-1), an important chemoattractant for monocytes. The objective of the present study was to examine the effect of Lp-X on MCP-1 expression in mesangial cells leading to an enhanced monocyte chemotaxis and to elucidate the mechanisms involved in this process. METHODS: Lp-X was isolated from the plasma of a patient with familial LCAT deficiency. After rat mesangial cells were incubated with Lp-X for four or six hours, the expression of MCP-1 mRNA was determined by nuclease protection assay, and MCP-1 protein was measured by Western immunoblotting analysis. Monocyte chemotaxis was determined by using a Micro Chemotaxis Chamber. RESULTS: Lp-X (50 to 100 nmol/mL) stimulated mesangial cell MCP-1 mRNA expression (137 to 220%) and MCP-1 protein levels (233 to 375%). Conditioned media collected from Lp-X-treated mesangial cells stimulated human acute monocytic leukemia (THP-1) monocyte chemotaxis (165 to 200%). The increase in MCP-1 expression in mesangial cells was associated with an elevation of intracellular diacylglycerol levels, and activation of protein kinase C (PKC) as well as nuclear factor-kappa B (NF-kappa B). CONCLUSION: These results suggest that Lp-X participates in the pathogenesis of glomerulosclerosis and subsequent renal failure in familial LCAT deficient patients by stimulating monocyte infiltration via a mechanism involving mesangial MCP-1 expression. (+info)
Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation.
(4/23)
Hypercholesterolemic human LDL contains oxidized subfractions that have atherogenic properties. Paradoxically, atherosclerosis incidence is low in patients with primary biliary cirrhosis (PBC), a disease characterized by marked increases in plasma LDL, including the LDL subfraction lipoprotein-X (Lp-X). To investigate the mechanisms underlying this paradox, we first examined the propensity to oxidation of unfractionated LDL isolated from PBC patients. After prolonged incubation with copper, PBC-LDL failed to increase the oxidation index or electrophoretic mobility noted in control LDL. An admixture of PBC-LDL or Lp-X with control LDL prevented oxidation of the latter in a dose-dependent manner. PBC-LDL was also noncompetitive against copper-oxidized LDL (oxLDL) for binding with a murine monoclonal anti-oxLDL antibody in a competitive ELISA. OxLDL exerts its proapoptotic and antiangiogenic effects in part by inhibiting fibroblast growth factor 2 (FGF2) expression. Preincubation of oxLDL with PBC-LDL, but not control LDL, attenuated the inhibitory effects of oxLDL on FGF2 expression in cultured bovine aortic endothelial cells (ECs). The antioxidant and prosurvival properties of PBC-LDL diminished after the patients underwent orthotopic liver transplantation. These results suggest that Lp-X reduces LDL atherogenicity by preventing LDL oxidation to protect EC integrity in the presence of hypercholesterolemia. They also suggest that altering LDL composition may be as important as reducing LDL concentration in preventing or treating atherosclerosis. (+info)
A novel in vivo lecithin-cholesterol acyltransferase (LCAT)-deficient mouse expressing predominantly LpX is associated with spontaneous glomerulopathy.
(5/23)
Complete lecithin cholesterol acyltransferase (LCAT) deficiency is a rare genetic cause of extreme reduction in high density lipoproteins and there is a high prevalence of chronic renal dysfunction that may progress to renal failure. Previous in vitro studies suggest the vesicular lipoprotein X (LpX) particles commonly seen in LCAT-deficient plasmas may be causative. To test this hypothesis, we have generated a novel murine model that selectively accumulate LpX in the circulation by cross breeding the sterol regulatory element binding protein (SREBP) 1a transgenic mice (S+) with the LCAT knockout (lcat-/-) mice. Fast protein liquid chromatography fractionation of pooled plasma lipids revealed that virtually all cholesterol is concentrated in the very low density lipoprotein (VLDL)-sized fractions. These fractions are enriched in free cholesterol and phospholipid but extremely poor in triglyceride. Electron microscopy of the d <1.063 g/ml fraction of the S+lcat-/- mice revealed abnormal large vesicular particles, suggestive of LpX. The S+lcat-/- mice developed glomerular lesions spontaneously evident at 6 months with glomerular and tubulointerstitial lipid-deposits. Immunohistochemical staining with RhoA showed marked positive focal staining in glomeruli in the S+lcat-/- mice and undetectable in the S+/lcat+/+ control. By 10 months of age, the kidneys showed progressive glomerular injury including segmental foam cell infiltrates, mesangial expansion, and hyalinosis. Renal abnormalities are very similar to those seen in human LCAT deficiency. We conclude that the selective high-level accumulation of plasma LpX in the S+lcat-/- mice is strongly associated with a spontaneous glomerulopathy, providing in vivo evidence that LpX contributes to the LCAT deficiency-related nephropathy. (+info)
Phospholipid transfer protein activity in two cholestatic patients.
(6/23)
CONTEXT: Plasma phospholipid transfer protein mediates the transfer of phospholipids from triglyceride-rich lipoproteins, very low density lipoproteins and low density lipoproteins to high density lipoproteins, a process that is also efficient between high density lipoprotein particles. It promotes a net movement of phospholipids, thereby generating small lipid-poor apolipoprotein AI that contains particles and subfractions that are good acceptors for cell cholesterol efflux. CASE REPORT: We measured the activity of plasma phospholipid transfer protein in two cholestatic patients, assuming that changes in activity would occur in serum that was positive for lipoprotein X. Both patients presented severe hypercholesterolemia, high levels of low density lipoprotein cholesterol and, in one case, low levels of high density lipoprotein cholesterol and high levels of phospholipid serum. The phospholipid transfer activity was close to the lower limit of the reference interval. To our knowledge, this is the first time such results have been presented. We propose that phospholipid transfer protein activity becomes reduced under cholestasis conditions because of changes in the chemical composition of high density lipoproteins, such as an increase in phospholipids content. Also, lipoprotein X, which is rich in phospholipids, could compete with high density lipoproteins as a substrate for phospholipid transfer protein. (+info)
Human lecithin:cholesterol acyltransferase deficiency: in vivo kinetics of low-density lipoprotein and lipoprotein-X.
(7/23)
OBJECTIVE: 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. (+info)
Improved specificity of a new homogeneous assay for LDL-cholesterol in serum with abnormal lipoproteins.
(8/23)
BACKGROUND: Although a homogeneous assay for serum LDL-cholesterol (LDL-C) has become a routine clinical procedure, problems remain in assay performance characteristics. METHODS: We examined the performance of a recently developed automated homogeneous assay (New-Daiichi assay) for serum LDL-C and compared the results with those obtained by the current homogeneous method (Denka-Seiken assay) or by ultracentrifugation as a control. RESULTS: The New-Daiichi assay showed satisfactory basic performance characteristics such as reproducibility, linearity, and stability. There was no interference in the assay by various substances examined. The LDL-C values obtained with this method correlated well with those obtained by ultracentrifugation. In samples from patients with obstructive jaundice, both methods detected cholesterol from abnormal lipoproteins (such as lipoprotein-X and -Y), but the New-Daiichi assay was less reactive and more specific for LDL-C. CONCLUSION: The new method has improved performance for the accurate measurement of LDL-C in clinical practice. (+info)