Proteolytic susceptibility of platelet low density lipoprotein receptor

In order to further characterize low density lipoprotein (LDL)-platelet interaction, we investigated the effect of protease pretreatment of human platelets on the subsequent binding of iodinated LDL (¹²⁵I-LDL). Our results showed that the platelet LDL receptor had a proteolytic susceptibility different from that of both classical LDL receptors and the fibrinogen receptor. Platelet pretreatment with chymotrypsin, trypsin, and pronase (at 50 μg/mL) had no effect on¹²⁵I-LDL binding, whereas fibroblast¹²⁵I-LDL binding was markedly reduced. Mild proteolytic digestion, however (up to 1 mg/mL), was helpful in characterizing the platelet LDL receptor. Scatchard analysis showed that chymotrypsin did not modify LDL binding characteristics, whereas trypsin and pronase altered maximal number of binding sites (B_max) without variation in dissociation constant. Trypsin increased B_max approximately twofold (2156±327 binding sites on control platelets vs. 5246±296 on treated platelets,P<0.001, mean±SEM, n=5), but pronase decreased B_max about 50% (2017±275 control vs. 1153±195 treated,P<0.001). A minimum of 30 min preincubation was required to detect significant effects, and apparent equilibrium was reached by 60 min. Maximal increase in platelet LDL binding sites induced by trypsin was observed at a protein concentration of 1 mg/mL at 37°C, whereas at 4°C no effect was found. In contrast, maximal pronase-inhibitory effect also was observed at 37°C but at higher protein concentration (10 mg/mL). Aprotinin, phenylmethylsulfonylfluoride, and soybean trypsin inhibitor were capable of fully blocking both the stimulation and the inhibition of platelet LDL binding induced by trypsin and pronase, respectively. Platelet pretreatment with both chymotrypsin and pronase (0.5 mg/mL) activated fibrinogen binding sites to a similar extent as ADP (100 μM). Furthermore, LDL (at a protein concentration of 0.3 mg/mL) increased by 81±6% the binding of fibrinogen to both protease- and ADP-stimulated platelets, but was unable to activate fibrinogen binding sites in unstimulated platelets. Over-all, the results suggest that platelet LDL receptor presents a different proteolytic susceptibility in comparison with both “classical” LDL receptor and fibrinogen receptor. Portions of this work were presented at the 17th Meeting of the European Lipoprotein Club, Tutzing, Germany, September 12–15, 1994.     

Growth modulation of low density lipoprotein receptor sterol sensitivity in cultured human fibroblasts

Sterols regulate low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expressions by end product repression. Studies on cultured cells have shown that growing cells have a higher LDL uptake than quiescent cells and that incubation of cells with growth factors or mitogenic compounds leads to sterol-resistant upregulation of LDL receptor gene expression. The recent finding that elevated LDL receptor activity in acute myelogenous leukemia cells was characterized by a decreased sensitivity to downregulation by sterols raises the possibility that the mechanism behind this is related to the cellular growth rate. By using cultured human fibroblasts as a model system we therefore studied whether growth modulation of sterol sensitivity takes place in normal actively growing cells. Judging from the ability of sterols (25-hydroxycholesterol+cholesterol) to inhibit ¹²⁵I-LDL degradation, we found that the sensitivity to sterols varied markedly between cells of different densities. The lowest sensitivity to sterols and highest ¹²⁵I-LDL degradation rate were found in subconfluent cells, whereas sparse and confluent cells were the most sensitive ones. In contrast to the LDL receptor, HMG-CoA reductase sterol sensitivity did not appear to be growth regulated. We conclude that growth-dependent modulation of sterol sensitivity and LDL receptor activity takes place in normal human fibroblasts. Modulation of sterol sensitivity may be an important mechanism to ensure an adequate cholesterol supply in growing cells.     

Upregulation of low density lipoprotein receptor activity by tumor necrosis factor,a process independent of tumor necrosis factor-induced lipid synthesis and secretion

It has been shown that tumor necrosis factor (TNF) rapidly upregulates expression of the low density lipoprotein (LDL) receptors on Hep G2 cells and acutely stimulates hepatic lipid synthesis and secretionin vivo. It may thus be possible that TNF-induced expression of LDL receptors is secondary to a decrease in cellular cholesterol content caused by TNF-stimulated lipid secretion. In order to know whether TNF upregulates LDL receptors by depletion of the cellular cholesterol content, the present experiments were designed to study the temporal relationship between TNF-stimulated expression of LDL receptor activity and TNF-induced changes in lipid synthesis and secretion in anin vitro setting by using Hep G2 cells (a highly differentiated human hepatoma cell line) as a hepatocyte model. Hep G2 cells were incubated with TNF (usually 2.5 nmol/L) for certain periods, and LDL receptor activity was evaluated by measuring [¹²⁵I]LDL binding at 4°C; lipid synthesis and secretion were assayed by measuring [³H]glycerol incorporation into triglycerides and phospholipids as well as [¹⁴C]acetate incorporation into cholesterol. We found that a 30-h exposure of the cells to TNF was needed for the effect of TNF to be seen on lipid synthesis and secretion as measured by incorporation of [³H]glycerol into triglycerides and phospholipids, whereas TNF rapidly (in several hours) upregulated LDL receptor activity. TNF stimulated triglyceride synthesis, but did not stimulate phospholipid synthesis. On the other hand, TNF stimulated phospholipid secretion, but did not stimulate triglyceride secretion. Exposure of the cells to TNF for 16 or 24 h neither decreased cholesterol synthesis nor stimulated cholesterol secretion as measured by [¹⁴C]acetate incorporation into cholesterol. Upregulation of LDL receptor activity through inhibition of cellular cholesterol synthesis with compactin (a competitive inhibitor of the 3-hydroxyl-3-methylglutaryl-CoA reductase) was augmented by TNF, whereas downregulation of LDL receptor activity through stimulation of cellular cholesterol synthesis with mevalonolactone almost completely blocked the upregulatory effect of TNF. In conclusion, TNF-stimulated expression of LDL receptor activity is not secondary to a depletion of cellular cholesterol content through TNF-stimulated lipid secretion or inhibition of cholesterol synthesis.     

Vitamin E reduces cholesterol esterification and uptake of acetylated low density lipoprotein in macrophages

The effects of vitamin E on cholesteryl ester (CE) metabolism in 1774 cells were examined. Pretreatment of 1774 cells with vitamin E at concentrations above 50 μM significantly decreased acetylated low density lipoprotein (LDL)-induced incorporation of [¹⁴C]oleate into CF in cells in a dose-dependent manner. This was partly due to vitamin E Also significantly inhibiting the uptake of [³H]CE-labeled acetylated LDL by 1774 cells. A trend existed toward suppression of acyl-CoA:cholesterol acyltransferase (ACAT) activity in the cell lysate at high vitamin E concentration, but there was no effect on hydrolysis of CE. These data indicate that vitamin E reduces the uptake of modified LDL and suppresses ACAT activity, resulting in less cholesterol esterification in macrophages; a novel mechanism underlying the antiatherogenic properties of vitamin E.     

Factors affecting resistance of low density lipoproteins to oxidation

Oxidation resistance (OR) of low density lipoproteins (LDL) is frequently determined by the conjugated diene (CD) assay, in which isolated LDL is exposed to Cu²⁺ as prooxidant in the range of 1–10 μM. A brief review on major findings obtained with this assay will be given. A consistent observation is that vitamin E supplements or oleic acid-rich diets increase OR. Oxidation indices measured by the CD assay and effects of antioxidants very significantly depend on the Cu²⁺ concentration used for LDL oxidation. For medium and high Cu²⁺ concentrations, the relationship between lag time and propagation rate can be described by a simple hyperbolic saturation function, which has the same mathematical form as the Michaelis-Menten equation. At medium and high Cu²⁺ concentrations (0.5 to 5 μM), vitamin E increases lag time in a dose-dependent manner. The increase is higher for 0.5 μM Cu²⁺ as compared to 5 μM. At low Cu²⁺ concentrations (0.5 μM or less), the mechanism of LDL oxidation changes. Significant oxidation occurs in a preoxidation phase, which commences shortly after addition of Cu²⁺. Preoxidation is not inhibited by vitamin E. It is concluded that much additional work is needed to validate the importance of oxidation indices derived from CD and similar assays.     

Quantitative determination of low density lipoprotein oxidation by FTIR and chemometric analysis

This study was conducted to develop a quantitative FTIR spectroscopy method to measure LDL lipid oxidation products and determine the effect of oxidation on LDL lipid and protein. In vitro LDL oxidation at 37°C for 1 h produced a range of conjugated diene (CD) (0.14–0.26 mM/mg protein) and carbonyl contents (0.9–3.8 μg/g protein) that were used to produce calibration sets. Spectra were collected from the calibration set and partial least squares regression was used to develop calibration models from spectral regions 4000-650, 3750-3000, 1720-1500, and 1180-935 cm⁻¹ to predict CD and carbonyl contents. The optimal models were selected based on their standard error of prediction (SEP), and the selected models were performance-tested with an additional set of LDL spectra. The best models for CD prediction were derived from spectral regions 4000-650 and 1180-935 cm⁻¹ with the lowest SEP of 0.013 and 0.013 mM/mg protein, respectively. The peaks at 1745 (cholesterol and TAG ester C=O stretch), 1710 (carbonyl C-O stretch), and 1621 cm⁻¹ (peptide C=O stretch) positively correlated with LDL oxidation. FTIR and chemometrics revealed protein conformation changes during LDL oxidation and provided a simple technique that has potential for rapidly observing structural changes in human LDL during oxidation and for measuring primary and secondary oxidation products.     

Abnormal suppression of 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured human fibroblasts by hypertriglyceridemic very low density lipoprotein subclasses

Our previous studies showed that hypertriglyceridemic very low density lipoproteins (HTG VLDL) are functionally abnormal. HTG VLDL, but not normal VLDL, suppress HMG-CoA reductase in cultured normal human fibroblasts. To determine if the suppression by HTG VLDL resulted from a subpopulation of smaller suppressive particles, more homogeneous subclasses of VLDL-VLDL₁ (S_f 100–400), VLDL₂ (S_f 60–100), and VLDL₃ (S_f 20–60) were obtained from the d<1.006 (g°ml⁻¹) fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient and tested for suppression in normal human fibroblasts. VLDL₁ and VLDL₂ from each of the 12 normolipemic subjects tested failed to suppress HMG-CoA reductase activity in normal fibroblasts. Eleven out of 12 preparations of normal VLDL₃ suppressed HMG-CoA reductase, but only one-third as effectively as LDL. By contrast, the VLDL₁, VLDL₂ and VLDL₃ from 15 out of 17 hypertriglyceridemic patients (hyperlipoproteinemia Types IIb, III, IV and V) were highly effective in suppression, with half-maximal suppression at 0.1–2.0 μg VLDL protein/ml. The VLDL abnormality is apparently associated with hypertriglyceridemia and not hypercholesterolemia, since VLDL from a homozygous familial hypercholesterolemia patient with a Type IIa pattern did not suppress whereas each of the VLDL subclasses from a Type IIb patient suppressed. Suppression by HTG VLDL in normal cells is apparently a consequence of interaction of the protein portion of the VLDL with the specific LDL cell surface receptor since HTG VLDL₁ treated with 0.1 M 1,2-cyclohexanedione to block arginyl residues failed to suppress the enzyme. Moreover, hypertriglyceridemic S_f 60–400 VLDL failed to suppress HMG-CoA reductase activity in LDL receptor-negative fibroblasts. There were no consistent major compositional differences between comparable normal and hypertriglyceridemic VLDL subclasses which could account for differences in suppression. All VLDL subclasses from Type III subjects were enriched in cholesteryl esters and depleted in triglyceride, relative to the corresponding normal VLDL subclasses. However, Type IV and Type V VLDL subclasses were normal in this repect. We conclude from these studies that small particle diameter is not required for suppression, since HTG VLDL₁ and VLDL₂ which contained few, if any, small particles were effective in suppression. Presented as part of the symposium “Low Density and Very Low Density Lipoproteins” at the American Oil Chemists' Society meeting on May 2, 1979, in San Francisco.     

Inhibition of 12- and 15-lipoxygenase activities and protection of human and tilapia low density lipoprotein oxidation by I-Tiao-Gung (<Emphasis Type="Italic">Glycine tomentella</Emphasis>)

I-Tiao-Gung, Glycine tomentella, has been used extensively as a traditional herbal medicine to relieve physical pain, but its bioactivity has not been studied systematically. Ninety-five percent ethanol extracts of G. tomentella (GT-E) showed antioxidant activity in human plasma by prolonging the lag phase (+T_lag) of Cu²⁺-induced, LDL oxidation and were dose dependent. The+T_lag of LDL combined with 3.2 μg/mL GT-E was similar to that with 2.0 μM (ca. 0.5 μg/mL) Trolox. A similar inhibitory effect was found toward tilapia plasma LDL. In addition, GT-E inhibited tilapia thrombocyte (nucleated platelet), 5-, 12-, and 15-lipoxygenase (LOX). The IC₅₀ values were 0.43, 0.72, and 0.42 μg/mL, respectively, whereas the IC₅₀ values for nordihydroguaiaretic acid (NDGA) on 5-, 12-, and 15-LOX were 2.3, 1.6, and 1.7 μg/mL, respectively. The IC₅₀ value for cyclooxygenase-2 (COX-2) inhibition by GT-E was 42.0 μg/mL, whereas the IC₅₀ value by indomethacin as a positive control was 0.61 μg/mL. The prevention of LDL oxidation and the dual inhibition of LOX and COX-2 are indicative of the possible roles of I-Tiao-Gung in antiatherosclerosis and anti-inflammation.     

Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines

Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990)Cancer Res. 50, 632–636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by¹⁴C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW1417, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in subconfluent cells. Evaluation of LDL receptor activity through¹²⁵I-LDL binding and internalization studies demonstrated LDL receptor expression was reduced by 37% in normal density cells relative to the low density cultures. In contrast to cholesterol synthesis, exogenous LDL could inhibit LDL receptor activity at both densities. Thus subconfluent growing colonic adenoCA cell lines retain the capacity for sterol repression, but, in contrast to normal fibroblasts, exhibit a high endogenous cholesterol synthesis which LDL cannot regulate.     

Dialysis and gel filtration of isolated low density lipoproteins do not cause a significant loss of low density lipoprotein tocopherol and carotenoid concentration

The resistance of isolated low density lipoprotein (LDL) to copper-initiated oxidation is often used as a measure of effectiveness of an antioxidant intervention. Prior to oxidation excess salt and EDTA are removed via dialysis or gel filtration of the LDL sample. However, there is concern over whether the antioxidant content of dialyzed or gel-filtered LDL is truly representative of native LDL extracted from a blood sample. Previously, the experiments done after the storage of native and dialyzed LDL at −80°C showed that the dialysis step can cause a loss of up to 60% in the tocopherol and carotenoid content of LDL. In the present study, a comparison of the micronutrient concentration in freshly prepared dialyzed and native LDL from 35 subjects showed that after the correction for cholesterol, only lycopene (13%, P<0.001) and to a lesser extent α-carotene (8%, P<0.02) were significantly decreased, and the absolute fall in concentration was far smaller than previously reported. Other experiments done with smaller numbers of samples suggested that there were minimal micronutrient losses following gel filtration and that it was important to include 10 μmol/L EDTA in the dialysis and elution buffer; otherwise micronutrient losses did occur. In summary, immediate dialysis of freshly isolated LDL in the presence of 10 μmol/L. EDTA does not cause any major loss in the concentration of tocopherol and most carotenoids.     

Human low density lipoprotein structure: Correlations with serum lipoprotein concentrations

Human low density lipoproteins (LDL) were isolated and purified from individuals having widely differing serum lipid concentrations. Very low density lipoproteins (VLDL) and high density lipoproteins (HDL) were also isolated and quantitated. HDL₂ and HDL₃ were separated by flotation velocity in the analytical ultracentrifuge and their relative weight percent determined. The mean density of LDL from 41 individuals was determined by flotation velocity at two different solvent densities. The mean density of LDL was directly proportional to the triglyceride (r=0.65) and VLDL (r=0.50) concentrations and inversely proportional to the HDL (r=−0.55) and HDL₂ (r=−0.74) concentrations (all significant at P<0.001). The mean molecular weight of LDL from 42 individuals was determined by flotation equilibrium centrifugation. The mean molecular weight of LDL was directly proportional to the HDL (r=0.49) and HDL₂ (r=0.48) concentrations and inversely proportional to the serum triglyceride (r=−0.60) and VLDL (r=−0.48) concentrations (all significant at P<0.005 except triglyceride—P<0.001). The molecular weight of LDL was inversely proportional to its density, and thus inversely proportional to its protein/lipid ratio which was confirmed by composition measurements. The density and molecular weight of LDL had no relationship to the concentration of LDL (r=0.04 and 0.03). A preliminary report of this study was given at the American Society for Biological Chemists Meeting in St. Louis, June 1981.     

Lipoprotein receptor mediated metabolism of [14C]arachidonic acid labeled chylomicron remnants by Hep G2 cells

During lipolysis of chylomicron triacylglycerol by lipoprotein lipase, arachidonic acid (AA) esters are hydrolyzed at a slower rate than the predominant 16–18 carbon fatty acid esters. The further metabolism of the AA that is hereby enriched in the chylomicron remnant acylglycerols has not been investigated. In the present study, we examined the low density lipoprotein (LDL) dependent and independent metabolism of [¹⁴C]AA present in chylomicron remnants in the human hepatoma cell line Hep G2. Mesenteric duct cannulated rats were fed [¹⁴C]AA and [³H]cholesterol in corn oil, and the chyle obtained was injected intravenously into hepatectomized rats to form chylomicron remnants labeled with [¹⁴C]AA in the triacylglycerol (TG) and with³H in the cholesteryl ester portion. The remnants were then incubated with Hep G2 cells. The uptake of [¹⁴C]AA within 2–4 h was similar to that of [³H]cholesteryl ester. After uptake into the cells, [¹⁴C]AA was preferentially incorporated into phospholipids, a high proportion being found in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. [¹⁴C]AA and [³H]cholesteryl ester uptake were influenced to similar extents by factors unknown to regulate the LDL receptor and by an anti-LDL receptor antibody. Addition of compactin thus increased the uptake of [¹⁴C]AA by 50% in 4 h and mevalonolactone decreased the uptake by 86%. Using an anti-LDL receptor antibody, 25.0% of [³H]cholesterol/cholesteryl ester and 37.7% of [¹⁴C]AA binding to the cells at 4°C were blocked. There was no lipolysis of [¹⁴C]TG or [¹⁴C]diacylglycerol by lipase secreted into the medium during incubations. The study shows that after the uptake of chylomicron remnants by Hep G2 cells, which in part occursvia the LDL receptor, AA is liberated from the acylglycerols and is preferentially incorporated into phospholipids.     

Plasma kinetic behavior in hyperlipidemic subjects of a lipidic microemulsion that binds to low density lipoprotein receptors

It was previously reported that a protein-free microemulsion (LDE) with structure roughly resembling that of the lipid portion of low density lipoprotein (LDL) was presumably taken up by LDL receptors when injected into the bloodstream. In contact with plasma, LDE acquires apolipoproteins (apo) including apo E that would be the ligand for receptor binding. Currently, apo were associated to LDE by incubation with high density lipoprotein (HDL). LDE-apo uptake by mononuclear cells showed a saturation kinetics, with an apparent K _m of 13.1 ng protein/mL. LDE-apo is able to displace LDL uptake by mononuclear cells with a K _i of 11.5 ng protein/mL. LDE without apo is, however, unable to displace LDL. The uptake of ¹⁴C-HDL is not dislocated by increasing amounts of LDE-apo, indicating that HDL and LDE-apo do not bind to the same receptor sites. In human hyperlipidemias, LDE labeled with ¹⁴C-cholesteryl ester behaved kinetically as expected for native LDL. LDE plasma disappearance curve obtained from eight hypercholesterolemic patients was markedly slower than that from 10 control normolipidemic subjects [fractional clearance rate (FCR)=0.02±0.01 and 0.12±0.04 h⁻¹, respectively; P<0.0001]. On the other hand, in four severely hypertriglyceridemic patients, LDE FCR was not significantly different from the controls (0.07±0.03 h⁻¹). These results suggest that LDE can be a useful device to study lipoprotein metabolism.     

Sedimentation equilibrium of human low density lipoprotein subfractions

The molecular weights of low density lipoprotein (LDL) subfractions were determined precisely by meniscus depletion sedimentation equilibrium. Equilibrium speeds ranged from 9743 to 5896 rpm. The average molecular weights of various LDL subfractions of S_f ^o values 9.49, 7.94, 6.42, 5.17, and 3.71 determined by sedimentation equilibrium were 2.97×10⁶; 3.13×10⁶; 2.89×10⁶; 2.45 ×10⁶; and 2.61×10⁶ daltons, respectively; and their respective densities were 1.0267, 1.0306, 1.0358, 1.0422, and 1.0492 g/ml. Minimal hydrated molecular weights for these fractions determined by flotation velocity at 37,020 rpm were 2.57×10⁶; 2.37×10⁶; 2.09×10⁶; 1.94×10⁶; and 1.81 ×10⁶ daltons; whereas similar molecular weights determined at 52,640 rpm were 2.53×10⁶; 2.27 ×10⁶; 1.99×10⁶; 1.86×10⁶; and 1.74×10⁶ daltons for the respective LDL subfractions. Higher molecular weights of fractions 2 and 5 compared to their adjacent fractions 1 and 4 by sedimentation equilibrium are of great interest. The calculated frictional ratio f/f^o from sedimentation equilibrium and flotation velocity data ranges from 1.10 to 1.31, suggesting complexity and asymmetry of LDL subfraction molecules. There is also evidence that compressibility of LDL molecules may be different than that for the salt solution under high g-force. Assuming that redistributed LDL molecules at equilibrium under low g-force are spherical, it is possible that the shape of LDL molecules undergoing flotation velocity determinations may be distorted in high g-force conditions. Such distortion may be consistent with the high f/f^o values obtained and may also be a basis for structural rearrangement and/or lipoprotein degradation with prolonged preparative ultracentrifugation at high g-force and pressure.     

Partial specific volume and preferential hydration of low density lipoprotein subfractions

We have determined the partial specific volume ( ) for five low density lipoprotein (LDL) subfractions (n=5–7) and evaluated preferential hydration (n=2) for LDL subfraction 3 in normolipoproteinemic subjects in order to characterize these highly atherogenic components of the human plasma lipoprotein spectra. Values for at 1 g were determined by sixth place density measurements of the solvent and lipoprotein solutions and carbon, hydrogen and nitrogen (CHN) absolute mass of the lipoprotein concentrations. Mean values for were 0.9757±0.0019, 0.9701±0.0007, 0.9674±0.0016, 0.9616±0.0016 and 0.9550±0.0025 ml/g for subfractions 1, 2, 3, 4 and 5, respectively. However, molecular densities (σ) obtained from ϱ(rho)=1/ for respective LDL subfractions were 1.0249, 1.0308, 1.0337, 1.0399 and 1.0471 g/ml, respectively. The preferential hydration of lipoprotein subfraction 3 (n=2) in NaCl/H₂O solutions was 2.9–4.8 wt percent, whereas values were much lower (0.3–0.6 wt percent) in NaCl/NaBr/H₂O solvent system. Unhydrated densities for LDL subfraction 3 (n=2) at 1 g (sixth-place density meter) were 1.0287 and 1.0269 g/ml, whereas at 200,000 × g (used in D₂O flotation ηF^o vs ϱ determinations) both values were 1.0308 g/ml, indicating that these similar LDL fractions have 23 and 53% higher compressibility than the solvent at 200,000 × g force. It was observed that the linearity of ηF^o vs ϱ may not be valid for solvents NaCl/NaBr/H₂O of density as high as 1.4744 g/ml. Thus, flotation velocity data using extreme salt concentrations (1.4744 g/ml and higher) may be viewed with caution.     

Low concentration of oxidized low density lipoprotein suppresses platelet reactivity <Emphasis Type="Italic">in vitro</Emphasis>: An intracellular study

The intracellular mechanisms underlying oxidized low density lipoprotein (oxLDL)-signaling pathways in platelets remain obscure and findings have been controversial. Therefore, we examined the influence of oxLDL in washed human platelets. In this study oxLDL concentration-dependently (20–100 μg/mL) inhibited platelet aggregation in human platelets stimulated by collagen (1 μg/mL) and arachidonic acid (60 μM), but not by thrombin (0.02 U/mL). The activity of oxLDL was greater at 24 h in inhibiting platelet aggregation than at 12 h. At 24 h, oxLDL concentration-dependently inhibited intracellular Ca²⁺ mobilization and thromboxane B₂ formation in human platelets stimulated by collagen. In addition, at 24 h oxLDL (40 and 80 μg/mL) significantly increased the formation of cyclic AMP, but not cyclic GMP or nitrate. In an ESR study, 24 h-oxLDL (40 μg/mL) markedly reduced the ESR signal intensity of hydroxyl radicals (OH⁻) in both collagen (2 μg/mL)-activated platelets and Fenton reaction (H₂O₂+Fe²⁺). The inhibitory effect of oxLDL may induce radical-radical termination reactions by oxLDL-derived lipid radical interactions with free radicals (such as hydroxyl radicals) released from activated platelets, with a resultant lowering of intracellular Ca²⁺ mobilization followed by inhibition of thromboxane A₂ formation, thereby leading to increased cyclic AMP formation and finally inhibited platelet aggregation. This study provides new insights concerning the effect of oxLDL in platelet aggregation.     

Ezetimibe Inhibits Expression of Acid Sphingomyelinase in Liver and Intestine

Ezetimibe inhibits cholesterol absorption in the intestine. Sphingomyelin has strong interactions with cholesterol. We investigated the effects of ezetimibe on Sphingomyelinase (SMase) expression in intestine and liver. After feeding rats with ezetimibe (5 mg/kg per day) for 14 days, acid SMase activities in the liver and in the proximal part of small intestine were reduced by 34 and 25%, respectively. Alkaline SMase (alk-SMase) was increased in the proximal part of the small intestine. Administration of lower doses of ezetimibe reduced acid SMase only in the liver by 14% (P < 0.05). In cell culture studies, ezetimibe decreased acid SMase activity in Hep G2 and Caco-2 cells dose-dependently. The reductions were more rapid for Hep G2 cells than for Caco-2 cells. Western blot showed that acid SMase protein was decreased in both Hep G2 and Caco-2 cells by 100 μM ezetimibe. The SM content was increased in Hep G2 cells but not Caco-2 cells, and total cholesterol content was increased in both cell lines 24 h after stimulation with 100 μM ezetimibe. Mevastatin, the inhibitor of cholesterol synthesis, induced a mild increase in acid SMase activity in Hep G2 cells but not Caco-2 cells. Following the reduction of acid SMase, ezetimibe at high dose slightly increased alk-SMase activity. In conclusion, the study demonstrates an inhibitory effect of ezetimibe on acid SMase activity and expression in both liver and intestine.     

Headspace gas chromatography to determine human low density lipoprotein oxidation

We previously described a rapid headspace gas chromatographic method for the determination of hexanal, an important decomposition product of n−6 polyunsaturated fatty acid (PUFA) peroxidation in rat liver samples and human red blood cell membranes. This method was applied to the measurement of Cu²⁺ catalyzed-oxidation of freshly prepared human low density lipoproteins (LDL) from 10 healthy adult volunteers. A twofold variation in oxidative susceptibility was found by this assay for hexanal and other volatiles. Hexanal values correlated significantly (P<0.05) with total polyunsaturated fatty acid (PUFA), 18∶2 and n−6 PUFA contents of LDL; but poorly with 20∶4 and with vitamin E. Therefore, in addition to α-tocopherol, other endogenous antioxidants and factors may contribute, to LDL's resistance to oxidation. This simple, rapid and sensitive method for oxidative susceptibility provides a useful component in the analysis of the prooxidant/antioxidant status of biological samples. The method is used routinely in our laboratories to determine specific peroxidation products of n−6 and n−3 PUFA.     

Detection of oxysterols in oxidatively modified low density lipoprotein by MALDI‐TOF MS

A simple method for the detection of oxysterols in oxidatively modified LDL (Ox‐LDL) has been developed using MALDI‐TOF MS. To identify the ion peaks of oxysterols, seven major oxysterols in Ox‐LDL (7α‐hydroxycholesterol, 7β‐hydroxycholesterol, 7‐ketocholesterol, 5α,6α‐epoxycholesterol, 5β,6β‐epoxycholesterol, 25‐hydroxychokesterol, (25R)‐26‐hydroxycholesterol), and cholesta‐3,5‐dien‐7‐one were analyzed by MALDI‐TOF MS. Among these oxysterols, 7‐ketocholesterol, a very abundant oxysterol in Ox‐LDL, was found to show a characteristic peak of [M + H]⁺ at m/z 401. Cholesta‐3,5‐dien‐7‐one, which is known as a degradation product of 7‐ketocholesterol upon saponification of Ox‐LDL, gave a major peak of [M + H]⁺ at m/z 383. In contrast, other oxysterols showed similar peak patterns at m/z 367 and 385. These results were applied to the analysis of Ox‐LDL by MALDI‐TOF MS after saponification and hexane‐extraction, detecting ion peaks at m/z 367, 383, 385, and 401. This MALDI‐TOF MS method has a potential as a simple tool to show the presence of oxysterols in Ox‐LDL without derivatization and chromatographic separation.     

Enhancement of sterol synthesis by the monoterpene perillyl alcohol is unaffected by competitive 3-hydroxy-3-methylglutaryl-CoA reductase inhibition

Monoterpenes such as limonene and perillyl alcohol (PA) are currently under investigation for their chemotherapeutic properties which have been tied to their ability to affect protein isoprenylation. Because PA affects the synthesis of isoprenoids’ such as ubiquinone’ and cholesterol is the end product of the synthetic pathway from which this isoprenoid pathway branches’ we investigated the effects of this compound upon cholesterol metabolism in the colonic adenocarcinoma cell line SW480. PA (1 mM) inhibited incorporation of ¹⁴C-mevalonate into 21–26 kDa proteins by 25% in SW480 cells. Cholesterol (CH) biosynthesis was assessed by measuring the incorporation of ¹⁴C-acetate and ¹⁴C-mevalonate into 27-carbon-sterols. Cells treated with PA (1 mM) exhibited a fourfold increase in the incorporation of ¹⁴C-acetate but not ¹⁴C-mevalonate into cholesterol. Mevinolin (lovastatin)’ an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase’ at 2 μM concentration’ inhibited CH synthesis from ¹⁴C-acetate by 80%. Surprisingly’ concurrent addition of mevinolin and PA did not significantly alter the stimulatory effects of PA. As observed differences in ¹⁴C-acetate and ¹⁴C-mevalonate precursor labeling could indicate PA affects early pathway events’ the effects of this monoterpene on HMG-CoA reductase activity were evaluated. Unexpectedly’ 1 mM PA did not stimulate activity of this enzyme. Consistent with its action as a reversibly bound inhibitor’ in washed microsomes’ 2 μM mevinolin pretreatment increased reductase protein expression causing a 12.7 (±2.4)-fold compensatory HMG-CoA reductase activity increase; concurrent treatment with 1 mM PA attenuated this to a 5.3 (±0.03)-fold increase. Gas chromatographic analysis confirmed CH was the major lipid present in the measured thin-layer chromatography spot. Since ¹⁴C-acetate incorporation into free fatty acid and phospholipid pools was not significantly affected by PA treatment’ nonspecific changes in whole acetate pool sizes were not indicated. Because increases in endogenous CH synthesis should result in compensatory changes in exogenous sterol utilization’ the effects of PA upon low density lipoprotein (LDL) receptor activity were evaluated. Consistent with the observed increases in CH synthesis’ 1 mM PA decreased ¹²⁵I-LDL internalization to 50% of the fetal bovine serum control; concurrent addition of 2 μM mevinolin attenuated this effect to a reduction of 80% of the control value. Data suggest that in certain colonic tumor cells PA strongly affects cholesterol metabolism via a mechanism of action that is insensitive to the HMG-CoA reductase inhibitor mevinolin.