Normal Human Low Density Lipoprotein Research Articles

Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine.

The "French paradox" (apparent compatibility of a high fat diet with a low incidence of coronary atherosclerosis) has been attributed to the regular drinking of red wine. However, the alcohol content of wine may not be the sole explanation for this protection. Red wine also contains phenolic compounds, and the antioxidant properties of these may have an important role. In in-vitro studies with phenolic substances in red wine and normal human low-density lipoprotein (LDL) we found that red wine inhibits the copper-catalysed oxidation of LDL. Wine diluted 1000-fold containing 10 mumol/L total phenolics inhibited LDL oxidation significantly more than alpha-tocopherol. Our findings show that the non-alcoholic components of red wine have potent antioxidant properties toward oxidation of human LDL.

Assessment of 5-lipoxygenase involvement in human monocyte-mediated LDL oxidation.

Lipoxygenase (LO) activity has been implicated in the process by which activated human monocytes oxidize normal human low density lipoprotein (LDL) and render it toxic to target cells. Here we examined the role of 5-LO in activated monocyte-mediated LDL modification. Five putative inhibitors of 5-LO (A63162, CGS8515, PF5901, RG6866, and MK886) were used to determine if they prevented activated monocytes from oxidizing LDL. Only RG6866, A63162, and CGS8515 inhibited monocyte-mediated LDL oxidation. Nonspecific effects of these drugs on LDL oxidation by activated monocytes were examined. RG6866 and A63162 were both found to be general antioxidants at their effective concentrations. CGS8515 was toxic at its effective concentration. A63162, CGS8515, and RG6866 also inhibited 15-LO activity in vitro. MK886 and PF5901 did not exhibit the nonspecific effects above and did not inhibit monocyte-mediated LDL oxidation, whereas both MK886 and PF5901 inhibited production of 5-LO metabolites by activated monocytes at concentrations that had no effect on LDL oxidation by the activated monocytes. Since neither of these agents inhibited LDL oxidation, we conclude that 5-LO is not involved in human monocyte oxidation of LDL. The possibility that a cellular 12- or 15-LO is involved in human monocyte-mediated LDL oxidation remains to be evaluated.

13C NMR evidence that substitution of glutamine for arginine 3500 in familial defective apolipoprotein B-100 disrupts the conformation of the receptor-binding domain.

Familial defective apoB-100 is a genetic mutation that is characterized by abnormal low density lipoprotein (LDL) and moderate hypercholesterolemia. Heterozygotes for this disorder possess two populations of LDL. One has normal receptor binding, and the other, which can be isolated by monoclonal antibody 19 immunoaffinity chromatography, has almost no binding activity. The mutation that disrupts binding is a Gln for Arg substitution of apoB-100 residue 3500. NMR spectra of LDL containing (13CH3)2Lys residues show that chemically modified Lys exist in two microenvironments. In normal human LDL, there are about 50 Lys with pK 8.9 and 170 Lys with pK 10.5; an upper limit of 10 pK 8.9 Lys may be particularly involved in binding to the LDL receptor. Examination of the mixture of normal LDL and mutant LDL from five patients shows that the latter have fewer pK 8.9 Lys. In purified defective LDL at least seven Lys are redistributed from the active to normal pool. The CD spectra of mutant and normal LDL are identical. Therefore, substitution of Gln for Arg at position 3500 induces a change in local conformation which disrupts the receptor-binding domain of apoB-100.

Lipoxygenase-mediated transformation of human low density lipoprotein to an oxidized and cytotoxic complex.

We have been studying the mechanisms involved in the oxidative modification of low density lipoprotein (LDL) that lead to its transformation to a cytotoxic complex. Here we examine the direct effect-of soybean lipoxygenase (SLO), a 15-lipoxygenase, on normal human LDL. SLO oxidized LDL and rendered it cytotoxic; agents known to interfere with lipoxygenase activity inhibited this reaction. Enhancement of both the SLO-mediated LDL oxidation and the conversion of LDL to a cytotoxin was observed when either superoxide dismutase or copper (II) (3,5,-diisopropylsalicylic acid)2, both of which dismute superoxide anion, were included during the incubation of SLO with LDL. In contrast, catalase inhibited this reaction in the presence or absence of agents that dismute superoxide anion. Thus, purified lipoxygenase can mediate LDL modification and superoxide anion inhibits this reaction, Furthermore, H2O2 is essential for SLO-mediated LDL oxidation and conversion of LDL to a cytotoxin.

Role of platelets in cholesteryl ester formation by U-937 cells.

The conversion of tissue macrophages into cholesteryl ester-rich foam cells is a crucial early event in atherogenesis. We studied the platelet as a potential source of cholesterol for esterification by macrophages because (a) platelets are rich in free cholesterol, (b) they adhere to macrophages early in atherogenesis, and (c) vascular injury can induce foam cell formation in the absence of hyperlipoproteinemia. We found that washed, activated human platelets from normocholesterolemic donors stimulated cholesteryl ester formation by the human monocyte-derived cell, U-937. Platelet cholesterol, released from platelets activated with calf skin collagen, was approximately equipotent at donating cholesterol to U-937 cells for esterification as normal human low density lipoprotein cholesterol. The stimulation of cholesteryl ester formation by activated human platelets demonstrated both concentration and time dependence. When hypercholesterolemic donors were studied, it was found that increasing plasma levels of cholesterol correlated directly with the ability of these hypercholesterolemic platelets to support cholesteryl ester synthesis by U-937 cells. Cholesterol-donating activity was also found in a 1,000-g supernatants of activated platelets. These observations point to a new and potentially important role for platelets in atherogenesis and suggest a mechanism for foam cell formation in the absence of marked hypercholesterolemia.

Physical properties of apoprotein B in mixed micelles with sodium deoxycholate and in a vesicle with dimyristoyl phosphatidylcholine.

Apoprotein B, the major apoprotein of normal human low density lipoprotein (LDL) was solubilized with sodium deoxycholate (NaDC). The protein was recombined with the phospholipid dimyristoyl phosphatidylcholine (DMPC) to produce a complex of DMPC-apoB (4:1 w/w). (Biochemistry. 22: 3170-3178. 1983). Carboxyfluorescein and [3H]dextran entrapment studies show the DMPC-apoB 4:1 (w/w) complex to encapsulate an aqueous volume of 0.17 microliter/mumol of DMPC. From the chemistry and morphology of the complex and the evidence that the complex possesses an encapsulated volume, the most appropriate structural model for this assembly is that of a phospholipid single bilayer vesicle into which apoB is incorporated. Differential scanning calorimetry (DSC) and circular dichroic spectroscopy (CD) were used to investigate the physical properties of apoB in the mixed micellar complex with NaDC and in the vesicular DMPC-apoB complex. CD studies of apoB in NaDC mixed micelles show that apoB exhibits a similar secondary structure as apoB of native LDL over the temperature range 5-30 degrees C. Reversible structural changes occur between 30 and 50 degrees C. However, above 50 degrees C, disruption of the micellar particle and endothermic protein unfolding and denaturation occur with a Tmax of 52 degrees C and an enthalpy of 0.22 cal/g apoB, as shown by DSC. The DMPC-apoB complex exhibits a reversible thermal transition centered at 24 degrees C (delta H = 3.34 Kcal/mol DMPC) which is associated with the order-disorder transition of the hydrocarbon chains of DMPC. An endothermic transition occurs over the range 53-70 degrees C (delta H = 2.09 cal/g apoB) which, as shown by CD and turbidity study, corresponds to protein unfolding-denaturation and particle disruption. CD shows that apoB in the vesicular environment undergoes a series of conformational changes. The major alterations occur over the temperature range of the order-disorder transition of the phospholipid. Between 37-60 degrees C, the conformation is similar to that observed in native LDL.

Rapid method for determining cholesteryl ester transitions of apoB-containing lipoproteins.

A wide variety of cholesteryl ester-rich apoB-containing lipoproteins undergo an order-disorder transition in the cholesteryl ester core at approximately normal body temperature. The transition occurs over several degrees C with the mid-point being as high as 57 degrees C in some cholesterol-fed animals. The transition mid-point of normal human low density lipoprotein (LDL) appears to vary from as low as 26 degrees C to about body temperature. However, to screen a large population of patients at risk for atheroscerlotic cardiovascular disease (ACD), a rapid method for determining the transition temperature of LDL is needed. Since apoB-containing lipoproteins (VLDL and LDL) are readily precipitated from plasma by dextran sulfate and magnesium sulfate, we have studied the thermal properties of this precipitate using differential scanning calorimetry (DSC). The VLDL-LDL precipitate undergoes a reversible thermal transition similar in transition temperature and enthalpy to the cholesteryl ester transition of isolated pure LDL. The transition is seen with the precipitate from VLDL-free plasma, but no transition is seen when VLDL and LDL have been removed. Cholesteryl ester-rich apoB containing lipoproteins were isolated from a variety of sources (man, cholesterol-fed monkeys, and rabbits) and their transition temperatures compared with the apoB-containing lipoprotein precipitates from the same source. The mid-point of individual transitions varied over a wide range (17-57 degrees C) and the correlation between the pure lipoprotein and the plasma precipitate was strong (r = 0.98, P < 0.001. Thus, DSC of the plasma apoB precipitate may be used as a rapid method of determining the cholesteryl ester transition of LDL and other apoB-containing lipoproteins.-Waugh, D. A., and D. M. Small. Rapid method for determining cholesteryl ester transitions of apoB-containing lipoproteins.