Protocol for efficient plasma sampling for low density lipoprotein turnover studies

Radiolabeled low density lipoprotein (LDL) is commonly used to study the turnover of LDL apolipoprotein B (apoB), the major protein component of LDL. Following an intravenous injection of radioiodinated LDL, typical sampling schedules have including 20-25 samples over a 14-day period with frequent sampling during the first 12 hr and daily samples thereafter. This is a burdensome task for subjects and investigators. To improve acceptance of the procedure, we have examined the effects of reduced sampling schedules upon the estimation of the fractional catabolic rate (FCR) for LDL apoB. Data from 36 different sets of LDL decay curves obtained from investigations of subjects with a variety of lipoprotein phenotypes have been used to test these schedules. Our results indicate that by choosing specific intervals over a 14-day period only 10 samples are sufficient to accurately determine the fractional catabolic rate for LDL in plasma. This reduced sampling schedule should facilitate the study of LDL turnover in large groups of subjects as outpatients.     

The sites of degradation of purified rat low density lipoprotein and high density lipoprotein in the rat

Low density lipoprotein and high density lipoprotein were isolated from rat serum by sequential ultracentrifugation in the density intervals 1.025-1.050 g/ml and 1.125-1.21 g/ml, respectively. The isolated lipoproteins were radioiodinated using ICl. Low density lipoprotein was further purified by concanavalin A affinity chromatography and concentrated by ultracentrifugation. 95% of the purified low density lipoprotein radioactivity was precipitable by tetramethylurea, while only 4% was associated with lipids. The radioiodinated high density lipoprotein was incubated for 1 h at 4 degrees C with unlabelled very low density lipoprotein, followed by reisolation by sequential ultracentrifugation. Only 3% of the radioactivity was associated with lipids and 90% was present on apolipoprotein A-I. The serum decay curves of labelled and subsequently purified rat low and high density lipoprotein, measured over a period of 28 h, clearly exhibited more than one component, in contrast to the monoexponential decay curves of iodinated human low density lipoprotein. The decay curves were not affected by the methods used to purify the LDL and HDL preparations. The catabolic sites of the labelled rat lipoproteins were analyzed in vivo using leupeptin-treated rats. In vivo treatment of rats with leupeptin did not affect the rate of disappearance from serum of intravenously injected labelled rat low density lipoprotein and high density lipoprotein. Leupeptin-dependent accumulation of radioiodine occurred almost exclusively in the liver after intravenous injection of iodinated low density lipoprotein, while both the liver and the kidneys showed leupeptin-dependent accumulation of radioactivity after injection of iodinated high density lipoprotein.     

Metabolic heterogeneity in the formation of low density lipoprotein from very low density lipoprotein in the rat: evidence for the independent production of a low density lipoprotein subfraction.

The formation of low density lipoprotein (LDL) from very low density lipoprotein (VLDL) was studied after injecting 14C-radiomethylated or 125I-radioiodinated VLDL into rats. VLDL and LDL B apoprotein specific radioactivity time curves were obtained after tetramethylurea extraction of the lipoproteins. In all experiments, the specific activity of LDL B apoprotein did not intercept the VLDL curve at maximal heights, suggesting that not all LDL B apoprotein is derived from VLDL B apoprotein. Further subfractionation of LDL into the Sf 12-20, 5-12, and 0-5 ranges showed that most (65%) LDL B apoprotein was present in the Sf 0-5 fraction and that only a small proportion (6-15%) of this fraction was derived from VLDL. However, the curves obtained for the Sf 12-20 and 5-12 subfractions were consistent with a precursor-product relationship in which all of these fractions were derived entirely from VLDL catabolism. These results contrasted strikingly with similar data obtained for normal humans in which all LDL is derived from VLDL. In the rat, it appears that most of the B apoprotein in the Sf 0-5 range, which contains 65% of the total LDL B apoprotein, enters the plasma independently of VLDL secretion.     

Comparison of plasma clearance of low density lipoprotein with beta-very low density lipoprotein or acetoacetylated low density lipoprotein in cholesterol-fed rabbits

The plasma clearance and tissue distribution of radioiodinated low-density lipoprotein (LDL), beta-very low density lipoprotein (beta-VLDL), and acetoacetylated LDL were studied in cholesterol-fed rabbits. Radioiodinated LDL ([125I]LDL) was cleared more slowly than either [125I]beta-VLDL or acetoacetylated-[125I]LDL and its fractional catabolic rate was one-half that of [125I]beta-VLDL and one-ninth that of acetoacetylated-[125I]LDL. Forty-eight hours after the injection of the labeled lipoproteins, the hepatic uptake was the greatest among the organs evaluated with the uptake of [125I]LDL being one-third that of either [125I]beta-VLDL or acetoacetylated-[125I]LDL. The reduction in the hepatic uptake of LDL due to a down-regulation of the receptors would account for this retarded plasma clearance.     

Guggulsterone enhances glycosylated low density lipoprotein binding in rat liver

Modification of low density lipoprotein by nonenzymic glycosylation resulted in decreased receptor-mediated lipoprotein catabolism. Guggulsterone treatment caused significant increase in binding of [¹²⁵I] low density lipoprotein as well as [¹²⁵I] glycosylated low density lipoprotein. Scatchard plot analysis of the binding activity revealed that under the influence of guggulsterone, the liver membrane contains increased amounts of a functional lipoprotein receptor that binds more low density lipoprotein particles.     

High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein (¹²⁵I) and in the cholesteryl ester (CE) moiety ([³H]). The metabolism of ¹²⁵I-/[³H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([³H]). In contrast, in LDLR^−/− mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR^−/− mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR^−/− mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.     

Is hypertriglyceridemic very low density lipoprotein a precursor of normal low density lipoprotein?

The precursor-product relationship of very low density (VLDL) and low density lipoproteins (LDL) was studied. VLDL obtained from normal (NTG) and hypertriglyceridemic (HTG) subjects was fractionated by zonal ultracentrifugation and subjected to in vitro lipolysis. The individual subfractions and their isolated lipolysis products, as well as IDL and LDL, were rigorously characterized. A striking difference in the contribution of cholesteryl ester to VLDL is noted. In NTG subfractions, the cholesteryl ester to protein ratio increases with decreasing density (VLDL-I----VLDL-III). This is the expected result of triglyceride loss through lipolysis and cholesteryl ester gain through core-lipid transfer protein action. In HTG subfractions there is an abnormal enrichment of cholesteryl esters that is most marked in VLDL-I and nearly absent in VLDL-III. Thus, the trend of the cholesteryl ester to protein ratios is reversed, being highest in HTG-VLDL-I and lowest in VLDL-III. This is incompatible with the precursor-product relationship described by the VLDL----IDL----LDL cascade. In vitro lipolysis studies support the conclusion that not all HTG-VLDL can be metabolized to LDL. While all NTG subfractions yield products that are LDL-like in size, density, and composition, only HTG-VLDL-III, whose composition is most similar to normal, does so. HTG VLDL-I and VLDL-II products are large and light populations that are highly enriched in cholesteryl ester. We suggest that this abnormal enrichment of HTG-VLDL with cholesteryl ester results from the prolonged action of core-lipid transfer protein on the slowly metabolized VLDL mass. This excess cholesteryl ester load, unaffected by the process of VLDL catabolism, remains entrapped within the abnormal particle. Therefore, lipolysis yields an abnormal, cholesteryl ester-rich product that can never become LDL.     

Self-assembled monolayer for low density lipoprotein detection

Heparin (HEP) has been covalently immobilized onto 4-aminothiophenol (ATP) self-assembled monolayer (SAM) deposited onto gold (Au)-coated glass plate for low density lipoprotein (LDL) detection. The HEP/ATP/Au and LDL/HEP/ATP/Au electrodes have been characterized using cyclic voltammetry (CV) and scanning electron microscopy (SEM). Surface plasmon resonance (SPR) measurements reveal that HEP/ATP/Au electrode is sensitive to detection of the LDL in the range 0.03 microM (10 mg/dl)-0.39 microM (130 mg/dl). The values of association and dissociation rate constants in the association phase calculated by kinetic analysis have been found to be k(a) = 9.67 x 10(1) M(-1) s(-1) and k(d) = 2.64 x 10(-4) s(-1).     

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein.

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the 'classical' scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.     

The low density lipoprotein receptor

The study of familial hypercholesterolemia at the molecular level has led to its advancement from a clinical syndrome to a fascinating experimental system. FH was first described 50 years ago by Carl Müller who concluded that the disease produces high plasma cholesterol levels and myocardial infarctions in young people, and is transmitted as an autosomal dominant trait determined by a single gene. The existence of two forms of FH, namely heterozygous and homozygous, was recognized by Khachadurian and Fredrickson and Levy much later. The value of FH as an experimental model system lies in the availability of homozygotes, because mutant genes can be studied without interference from the normal gene. The first and most important breakthrough was the realization that the defect underlying FH could be studied in cultured skin fibroblasts. Rapidly, the LDL receptor pathway was conceptualized and its dysfunction in cells from FH homozygotes was demonstrates. Isolation of the normal LDL receptor protein and studies on the biosynthesis and structure of abnormal receptors in mutant cell lines provided essential groundwork for elucidation of defects at the DNA level. The power of the experimental system, FH, became nowhere more obvious than in work that correlated structural information at the protein level with the elucidation of defined defects in the LDL receptor gene. In addition to revealing important structure-function relationships in the LDL receptor polypeptide and delineating mutational events, studies of FH have established several more general concepts. First, the tight coupling of LDL binding to its internalization suggested that endocytosis was not a non-specific process as suggested from early observations. The key finding was that LDL receptors clustered in coated pits, structures that had been described by Roth and Porter 10 years earlier. These investigators had demonstrated, in electron microscopic studies on the uptake of yolk proteins by mosquito oocytes, that coated pits pinch off from the cell surface and form coated vesicles that transport extracellular fluid into the cell. Studies on the LDL receptor system showed directly that receptor clustering in coated pits is the essential event in this kind of endocytosis, and thus established receptor-mediated endocytosis as a distinct mechanism for the transport of macromolecules across the plasma membrane. Subsequently, many additional systems of receptor-mediated endocytosis have been defined, and variations of the overall pathway have been described.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxidised low density lipoprotein causes human macrophage cell death through oxidant generation and inhibition of key catabolic enzymes

Oxidised low density lipoprotein (oxLDL) is thought to be a significant contributor to the death of macrophage cells observed in advanced atherosclerotic plaques. Using human-derived U937 cells we have examined the effect of cytotoxic oxLDL on oxidative stress and cellular catabolism.Within 3 h of the addition of oxLDL, there was a rapid, concentration dependent rise in cellular reactive oxygen species followed by the loss of cellular GSH, and the enzyme activity of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aconitase. The loss of these catabolic enzymes was accompanied by the loss of cellular ATP and lower lactate generation. Addition of the macrophage antioxidant 7,8-dihydroneopterin inhibited the ROS generation, glutathione loss and catabolic inactivation. NOX was shown to be activated by oxLDL addition while apocynin inhibited the loss of GSH and cell viability. The data suggests that oxLDL triggers an excess of ROS production through NOX activation, and catabolic failure through thiol oxidation resulting in cell death.     

Comparative studies of vertebrate lipoprotein lipase: a key enzyme of very low density lipoprotein metabolism

Lipoprotein lipase (LIPL or LPL; E.C.3.1.1.34) serves a dual function as a triglyceride lipase of circulating chylomicrons and very-low-density lipoproteins (VLDL) and facilitates receptor-mediated lipoprotein uptake into heart, muscle and adipose tissue. Comparative LPL amino acid sequences and protein structures and LPL gene locations were examined using data from several vertebrate genome projects. Mammalian LPL genes usually contained 9 coding exons on the positive strand. Vertebrate LPL sequences shared 58-99% identity as compared with 33-49% sequence identities with other vascular triglyceride lipases, hepatic lipase (HL) and endothelial lipase (EL). Two human LPL N-glycosylation sites were conserved among seven predicted sites for the vertebrate LPL sequences examined. Sequence alignments, key amino acid residues and conserved predicted secondary and tertiary structures were also studied. A CpG island was identified within the 5'-untranslated region of the human LPL gene which may contribute to the higher than average (×4.5 times) level of expression reported. Phylogenetic analyses examined the relationships and potential evolutionary origins of vertebrate lipase genes, LPL, LIPG (encoding EL) and LIPC (encoding HL) which suggested that these have been derived from gene duplication events of an ancestral neutral lipase gene, prior to the appearance of fish during vertebrate evolution. Comparative divergence rates for these vertebrate sequences indicated that LPL is evolving more slowly (2-3 times) than for LIPC and LIPG genes and proteins.     

Metabolism of apoE-free high density lipoproteins in rat hepatoma cells: evidence for a retroendocytic pathway

The cellular metabolism of apoE-free HDL (HDL) was studied in rat hepatoma cells (FU5AH). Cells incubated with HDL showed a dose-dependent decreased incorporation of [14C]acetate into cell sterol, indicating a net cholesterol delivery to the cells. HDL was localized both at the cell surface and inside the cell. This conclusion was drawn from both the association of 125I-labeled HDL with the cells under different experimental conditions and morphological evidence based on the association of colloidal gold-labeled HDL with the cells. Up to 63% of the 125I-labeled HDL protein initially inside the cell was subsequently recovered in the media as trichloroacetic acid precipitable (TCA-ppt) protein after a 30-min, 37 degrees C chase with a 100-fold concentration of unlabeled HDL. About 27% of the TCA-ppt apoprotein originally inside the cell was recovered as TCA-soluble material. Thus, we conclude that of the HDL apoprotein taken up by the cells, the majority is resecreted by a retroendocytosis pathway. The quantity of HDL apoprotein reappearing in the media was stimulated by the presence of unlabeled HDL in the media, while the amount of TCA-soluble material produced was not. Retroendocytosis of HDL was inhibited at 0 degree C and by the presence of 10 mM NaCN, 20 mM 2-deoxy-D-glucose in the media. Thus, the pathway appears to be both temperature- and energy-sensitive. HDL resecreted by the cell were depleted of cholesteryl ester and showed an altered size distribution, indicative of lipoprotein catabolism and remodeling. This study provides evidence for the existence of an endocytosis-retroendocytosis pathway for HDL apoproteins in a rat hepatoma cell and for the possibility that the endocytosis-retroendocytosis pathway may be involved in lipid delivery to the cell.     

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

Bile acids inhibit secretion of very low density lipoprotein by rat hepatocytes

The influence of taurocholate on very low density lipoprotein (VLDL) triacylglycerol synthesis and secretion was studied by isolated rat liver-parenchymal cells. The incorporation of [3H]glycerol into cell-associated and VLDL triacylglycerols were measured after incubation in medium containing 0.75 mM oleate. Taurocholate caused a maked decrease in VLDL [3H]triacylglycerol secretion from the hepatocytes: 50-150 microM taurocholate inhibited secretion of VLDL [3H]triacylglycerols by 70-90%. Similar results were obtained when the mass of secreted VLDL triacylglycerols was measured. Taurocholate caused a decreased secretion of VLDL [3H]triacylglycerols after 15-30 min incubation. A higher amount of cellular triacylglycerols was found in taurocholate-supplemented cells. Furthermore taurocholate did not change the intracellular lipolysis of triacylglycerols. These results suggest that bile acids interfere more probably with the assembly and/or secretion of VLDL-particles and not with earlier stages of VLDL formation, e.g. triacylglycerol synthesis.     

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.     

Interaction of rat plasma very low density lipoprotein with lipoprotein lipase-rich (postheparin) plasma.

Incubation of 125I-labeled very low density lipoprotein (VLDL) with lipoprotein lipase-rich (postheparin) plasma obtained from intact or supradiaphragmatic rats resulted in the transfer of more than 80% of apoprotein C from VLDL to high density lipoprotein (HDL), whereas apoprotein B was associated with lipoprotein of density less than 1.019 g/ml (intermediate lipoprotein). The transfer of 125I-labeled apoprotein C from VLDL to HDL increased with time and decreased in proportion to the amount of VLDL in the incubation system. A relationship was established between the content of triglycerides and apoprotein C in VLDL, whereas the amount of apoprotein C in VLDL was independent of that of other apoproteins, especially apoprotein B. The injection of heparin to rats preinjected with 125I-labeled VLDL caused apoprotein interconversions similar to those observed in vitro. The intermediate lipoprotein was relatively rich in apoprotein B, apoprotein VS-2, cholesterol, and phospholipids and poor in triglycerides and apoprotein C. The mean diameter of intermediate lipoprotein was 269 A (compared with 427 A, the mean Sf rate was 30.5 (compared with 115), and the mean weight was 7.0 X 10(6) daltons (compared with 23.1 X 10(6)). From these data it was possible to calculate the mass of lipids and apoproteins in single lipoprotein particles. The content of apoprotein B in both particles was virtually identical, 0.7 X 10(6) daltons. The relative amount of all other constituents in intermediate lipoprotein was lower than in VLDL: triglycerides, 22%; free cholesterol, 37%; esterified cholesterol, 68%; phospholipids, 41%; apoprotein C, 7%, and VS-2 apoprotein, 60%. The data indicate that (a) one and only one intermediate lipoprotein is formed from each VLDL particle, and (b) during the formation of the intermediate lipoprotein all lipid and apoprotein components other than apoprotein B leave the density range of VLDL to a varying degree. Whether these same changes occur during the clearance of VLDL in vivo is yet to be established.