Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity.

Small dense low density lipoprotein (LDL) particles have altered apolipoprotein (apo) B conformation and lowered affinity for the LDL receptor (J. Biol. Chem. 1994. 269: 511-519). Herein, we examine the interaction of small dense LDL with cell LDL receptor-independent binding sites. Compared to normal LDL, at low LDL cell media concentrations (<10 microg/ml), small dense LDL had decreased specific binding to the LDL receptor on normal fibroblasts at 4 degrees C, but a 2-fold increased binding to LDL receptor-independent cell sites. At higher LDL concentration (100 microg/ ml), LDL receptor-independent binding of small dense LDL was 4.5-fold that of normal LDL in normal fibroblasts, but greater (2- to 14- fold) in LDL receptor-negative fibroblasts. In LDL receptor-negative fibroblasts at 37 degrees C, small dense LDL had higher (3-fold) cell association than normal size LDL but no effective LDL degradation. At high LDL concentrations (> or =100 microg/ml), LDL binding to normal or LDL receptor-negative fibroblasts was not affected by several anti-apoB monoclonal antibodies or by cell pretreatment with proteases, chondroitinase, or neuraminidase. In contrast, pretreating normal and receptor-negative fibroblasts with heparinase and heparitinase decreased LDL cell binding by 35% and 50%, respectively. Similarly, preincubation of receptor-negative fibroblasts with sodium chlorate, an inhibitor of proteoglycan sulfation, decreased LDL binding by about 45%. We hypothesize that small dense LDL might be more atherogenic than normal size LDL due to decreased hepatic clearance by the LDL receptor, and enhanced anchoring to LDL receptor-independent binding sites in extrahepatic tissues (e.g., the arterial wall), a process mediated, in part, by cell surface proteoglycans.

Three-fold effect of lovastatin treatment on low density lipoprotein metabolism in subjects with hyperlipidemia: increase in receptor activity, decrease in apoB production, and decrease in particle affinity for the receptor. Results from a novel triple-tracer approach.

To differentiate effects of lovastatin on low density lipoprotein (LDL) receptor activity from effects on LDL metabolic properties, LDL apolipoprotein B (apoB) turnover was studied in eight hyperlipidemic subjects during baseline and lovastatin treatment, in the latter case with LDL tracers isolated during both baseline (CLDL) and drug treatment (Rx-LDL) conditions. Lovastatin (40 mg/day) significantly lowered total plasma and LDL cholesterol levels (27% and 25%, respectively) as well as plasma triglyceride levels (30%). Using contemporaneous tracers (C-LDL before and Rx-LDL during treatment), lovastatin caused a modest increase in LDL fractional catabolic rate (FCR) (0.410+/-0.113 vs. 0.339+/-0.108 pools/day, P < 0.04 by paired t). The increase in LDL tracer FCR was higher when C-LDL tracer isolated during the untreated period was injected during lovastatin treatment (0.496+/-0.177 vs. 0.339+/-0.108 pools/day, P < 0.02). These in vivo studies in humans were confirmed by injecting LDL tracers from two patients into five guinea pigs. The C-LDL tracer was cleared consistently faster than the Rx-LDL tracer (0.082+/-0.018 vs. 0.057+/-0.015 pools/h, P< 0.001). The results demonstrate three important outcomes of lovastatin treatment in these subjects: LDL receptor activity increased by 49% (P < 0.02); LDL apoB production rate decreased by 17% (P < 0.03), and LDL particle in vivo affinity for the LDL receptor decreased by 15% (P < 0.01). The decrease in LDL particle affinity partially negated the expected effect of increased LDL receptors on LDL clearance. The present study provides an explanation for earlier observations by several investigators using contemporaneous tracers that treatment with HMG-CoA reductase inhibitors resulted in only modest increases in low density lipoprotein functional catabolic rate.

Heparan sulfate proteoglycan-mediated uptake of apolipoprotein E-triglyceride-rich lipoprotein particles: a major pathway at physiological particle concentrations.

We explored potential mechanisms of non-low-density lipoprotein (LDL) receptor-mediated uptake of triglyceride-rich particles (TGRP) in the presence of apolipoprotein E (apo E). Human fibroblasts were incubated with model intermediate-density lipoprotein- (IDL-) sized TGRP (10-1000 microg of neutral lipid/mL) containing apo E. The extent of receptor-mediated uptake of TGRP was assessed with (a) an anti-apo E monoclonal antibody, which blocks receptor interaction; (b) incubation with heparin; (c) normal vs LDL receptor-negative fibroblasts; and (d) receptor-associated protein (RAP) to determine the potential contribution of LDL receptor-related protein (LRP). Cell surface heparan sulfate proteoglycan- (HSPG-) mediated uptake was examined with or without the addition of heparinase and heparitinase to cell incubation mixtures. At low particle concentrations (250 microg of neutral lipid/mL), most (>/=60%) particle uptake and internalization was via HSPG-mediated pathways. This HSPG pathway did not involve classical lipoprotein receptors, such as LRP or the LDL receptor. These data suggest that in peripheral tissues, such as the arterial wall, apo E may act in TGRP as a ligand for uptake not only via the LDL receptor and LRP pathways but also via HSPG pathways that are receptor-independent. Thus, at physiologic particle concentrations apo E-TGRP can be bound and internalized in certain cells by relatively low affinity but high capacity HSPG-mediated pathways.

Oxidized low density lipoproteins alter macrophage lipid uptake, apoptosis, viability and nitric oxide synthesis.

Uptake of oxidized low density lipoproteins (LDL) by monocyte macrophages to form "foam" cells occurs during formation of atherosclerotic lesions. Inducible nitric oxide synthase (iNOS) has been identified in foam cells. To investigate interactions between oxidized LDL, monocyte macrophage viability and iNOS, studies were performed with J774.Al macrophages. iNOS mRNA, protein and enzyme activity were induced in J774.Al macrophages by IFN-gamma lipopolysaccharide (LPS). Neither iNOS induction nor inhibition of nitric oxide (NO) formation was associated with significant alterations in the binding, uptake or degradation of native or oxidized LDL. Nontoxic doses of native LDL or of oxidized LDL did not influence iNOS mRNA or protein in macrophages. However, oxidized LDL, but not native LDL or acetyl LDL, inhibited NO production by macrophages in a dose- and time-dependent fashion. Inhibition of iNOS was not correlated with cholesteryl ester formation but with the degree of LDL oxidation. Inhibition of iNOS did not require the scavenger receptor or directed endocytosis and exhibited noncompetitive kinetics. Inhibition of iNOS activity in J774.Al macrophages was produced by lipid from oxidized LDL but not by lipid from native LDL and by PC vesicles containing LPC but not by PC vesicles alone. Inhibition of NO formation diminished apoptosis of the activated macrophages. The data suggest NO production by iNOS and inhibition of the enzyme by oxidized LDL lipid may influence cell viability, cell-cell interactions and vasomotor tone during atherogenesis.

Oleate and other long chain fatty acids stimulate low density lipoprotein receptor activity by enhancing acyl coenzyme A:cholesterol acyltransferase activity and altering intracellular regulatory cholesterol pools in cultured cells.

Modification of dietary fatty acid composition results in changes in plasma cholesterol levels in man. We examined the effect of in vitro fatty acid supplementation on low density lipoprotein (LDL) receptor activity in cultured cells and questioned whether changes were related to fatty acid-induced alterations in acyl-CoA: cholesterol acyltransferase (ACAT) activity. Preincubation of cultured cells (i.e. human skin fibroblasts, J774 macrophages, and HepG2 cells) with oleic acid (oleic acid:bovine serum albumin molar ratio 2:1) at 37 degrees C for longer than 2 h resulted in a 1.2- to 1.5-fold increase in LDL cell binding at 4 degrees C and LDL cell degradation at 37 degrees C. Scatchard analysis showed that oleic acid increased LDL receptor number but not LDL affinity (Kd). Fatty acid supplementation of J774 macrophages increased both LDL receptor activity and cholesteryl ester accumulation. The ACAT inhibitor, 58-035, eliminated both effects, and increased ACAT activity preceded stimulation of LDL receptor activity by 1-2 h. Supplementation of macrophages with triolein emulsion particles also increased LDL cell binding and degradation, and addition of cholesterol to the emulsions abolished this effect. Among fatty acids tested, oleate (18:1), arachidonate (20:4), and eicosapentanoate (20:5) demonstrated the greatest effects. We hypothesize that certain fatty acids delivered to cells either in free form, or as triglyceride, first increase cellular ACAT activity, which then causes a decrease in an intracellular free cholesterol pool, signaling a need for increased LDL receptor activity. This mechanism may play a role in the effect of certain dietary fatty acids on LDL metabolism in vivo.

Apoprotein B structure and receptor recognition of triglyceride-rich low density lipoprotein (LDL) is modified in small LDL but not in triglyceride-rich LDL of normal size.

We compared the effect of lipid composition and particle size of triglyceride-rich low density lipoprotein (LDL) upon apoprotein B conformation and binding to the LDL receptor. Three groups of triglyceride-rich LDL were studied: (a) LDL isolated from chronic hypertriglyceridemic individuals (HTG-LDL); (b) normal LDL made triglyceride-rich by in vitro incubation with triglyceride emulsion and the neutral lipid transfer protein (R-LDL); and (c) LDL from normolipidemic individuals made acutely hypertriglyceridemic by intravenous infusion of 10% Intralipid (IV-LDL). HTG-LDL was small and dense, whereas R-LDL and IV-LDL had normal size. HTG-LDL, but not R-LDL or IV-LDL, exhibited decreased binding to the LDL receptor on human skin fibroblasts in studies at 4 degrees C and reduced degradation at 37 degrees C. Apoprotein B conformation was assessed by circular dichroism and by analyzing the immunoreactivity of different monoclonal antibodies. HTG-LDL but not R-LDL or IV-LDL showed a change in the CD spectra and a consistent decrease in the immunoreactivity of monoclonal antibody 3F5 (2.5-fold) which recognizes an epitope adjacent to the receptor binding domain of apoprotein B. These findings suggest that in triglyceride-rich LDL, the relative content of neutral lipid in the core of LDL in the absence of changes in the size of the particle does not significantly affect apoprotein B conformation or its affinity for the LDL receptor.

Alterations in plasma alpha- and gamma-tocopherol concentrations in response to intravenous infusion of lipid emulsions in humans.

To study the fate of intravenously infused vitamin E, we infused lipid emulsions rich in gamma-tocopherol (Intralipid, Kabi, Stockholm, Sweden), or in both alpha- and gamma-tocopherols (Lipidem, Hausmann Laboratories, St Gallen, Switzerland); in normal human volunteers. Plasma gamma-tocopherol levels increased in four subjects infused with Intralipid 10% (0.3 g triglyceride [TG]/kg/h for 6 hours) from 3 +/- 1 to 25 +/- 2 nmol/mL, but by 24 hours they decreased to 5 +/- 1 nmol/mL. Although eight times more gamma-tocopherol was infused, plasma alpha-tocopherol levels also increased from 26 +/- 7 to 39 +/- 9 nmol/mL at 8 hours and decreased to 24 +/- 5 nmol/mL at 24 hours. Increases of alpha-tocopherol in the very-low-density lipoprotein (VLDL) density range occurred at 6 and 8 hours, while decreases occurred in low-density lipoprotein (LDL) and high-density lipoprotein (HDL) density ranges at 3, 6, 8, and 24 hours. Infusion of both emulsions in random order to six subjects at therapeutic rates (0.1 g/kg/h for 6 hours) resulted in (1) a threefold increase in plasma gamma-tocopherol concentrations at 6 hours, (2) increases in plasma alpha-tocopherol concentrations only with Lipidem (from 14.3 +/- 1.0 nmol/mL at 0 hours to 18.4 +/- 2.7 at 6 hours and 18.9 +/- 1.1 at 24 hours), and (3) no decreases in lipoprotein alpha-tocopherol levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins.

The ability to preserve low density lipoprotein (LDL) preparations frozen for weeks and months without changes in structure or biological properties is of potential use in long-term comparative studies of LDL. We demonstrate that freeze-thawing of LDL causes marked alterations in its structure and biological behavior, and that such changes can be prevented by the addition of sucrose to the LDL solution prior to freezing. Freezing LDL at -70 degrees C in the absence of sucrose resulted in aggregation and fusion of particles as measured by electron microscopy, spectrophotometric absorption, and column gel filtration. This was associated with increased binding affinity of monoclonal antibodies at epitopes distant from the receptor binding region. Functional changes induced by freezing included 3- to 10-fold increases in binding at 4 degrees C and 37 degrees C, and uptake of LDL in fibroblasts, attributable mainly to increases in nonspecific binding processes. Cryopreservation of LDL in 10% sucrose (w/v) completely prevented the structural and functional changes incurred after short-term freezing, and LDL cryopreserved in sucrose for as long as 18 months displayed cell binding, uptake, and degradation very similar to that of freshly obtained LDL.

Comparison of two special infant formulas designed for the treatment of protracted diarrhea.

The nutritional management of intractable diarrhea and short bowel syndrome remains a challenging problem. The advantage of continuous nasogastric infusion is undisputed, but what to feed remains in question, and no studies, to these authors' knowledge, have yet compared two widely used specially designed protein hydrolysate formulas. A randomized crossover trial of two periods of 7 days was carried out with Alfare and Pregestimil administered by a constant infusion pump in six malnourished infants aged 1-13 months. Two had intractable diarrhea, and four the short bowel syndrome. Identical quantities of calories were administered during the two periods. There was good tolerance for both formulas and satisfactory weight gain. Despite compositional differences related to osmolality, the source of the hydrolysates and their profile, and the qualitative pattern of the carbohydrates, no difference was observed with regard to stool weight, Na+ and K+ losses, and the enteral absorption of fat, carbohydrate-derived energy, and total energy. The percent absorption of nitrogen was somewhat higher (p less than 0.01) with Pregestimil (83.8 +/- 2.7) than with Alfare (77.3 +/- 3.4), but nitrogen retention was unaffected. Energy absorption was the same on both formulas, but maldigested or malabsorbed carbohydrates accounted for 63% of total energy loss during Alfare feeding and 72% during the week of Pregestimil. These data call for studies with protein hydrolysate formulas reformulated with a lower concentration of carbohydrates and a higher one of fat.

Taurine supplementation of a premature formula improves fat absorption in preterm infants.

The predominance of taurine (Tau) conjugated over glycine conjugated bile acids in infants fed human milk as opposed to those on formulas without added Tau could account for a more complete absorption of fat. Fifteen low birth weight infants were randomized to either Enfamil Premature or to Enfamil Premature added with 40 mumol/dl of Tau and compared to a third group made up of nine low birth weight infants fed their own mother's preterm milk. Formulas and human milk were fed according to tolerance and constituted the sole nutrition for 3 months. A metabolic study was carried out at 3 wk of age and control of growth was done periodically. Urinary Tau excretion (mumol/dl) was very low (p less than 0.001) in the group fed Enfamil Premature (0.3 +/- 0.1) when compared to the values obtained in infants supplemented with Tau (51.6 +/- 12.5) and in those on human milk (36.3 +/- 7.9). Infants supplemented with Tau (92.5 +/- 1.2) had a coefficient of fat absorption which was higher (p less than 0.05) than the unsupplemented group (87.5 +/- 7.9) and comparable to the human milk-fed group (91.6 +/- 1.4). The effect was more pronounced on the saturated fatty acids and varied inversely with their individual water solubility. There was no effect of Tau on nitrogen retention and growth was identical in the three groups. These data show that the addition of Tau to formula had no effect on growth but improved the absorption of fat especially saturated fatty acids which require higher concentrations of bile acids to form mixed micelles.