Low-density Lipoprotein Samples Research Articles

Alteration of plasma low density lipoprotein from smokers

Thiobarbituric acid reactive substances (TBARS) in 38 low density lipoprotein (LDL) samples from 21 healthy male non-smokers and 17 smokers (> 20 cigarettes per day) were measured before and after oxidation. TBARS in the freshly isolated LDL from non-smokers and smokers were similar, however, oxidized LDL samples from smokers developed nearly twofold more TBARS than non-smoker LDL samples. 16 LDLs from 8 smokers and 8 non-smokers were conditioned in redox-metal containing F-10 medium and subsequently added to P 388 D.1 macrophage cultures. LDL dependent cholesteryl ester increase in the P 388 D.1 cells after an 18 h incubation with non-smoker LDL was significantly lower than in the cells incubated with smoker LDL ( P < 0.01). A higher reacylation rate of cholesterol in P 388 D.1 cells incubated with smoker LDL ( P < 0.05) suggests that LDL-cholesterol uptake is significantly higher in P 388 D.1 cells incubated with smoker-LDL than in P 388 D.1 cultures exposed to non-smoker LDL. This finding indicates that smoking might contribute to increased shunting of LDL into macrophages. The vitamin E content of 6 non-smoker LDL samples was significantly higher than that in 6 smoker-LDL samples ( P < 0.01). We conclude that the vitamin E/LDL ratio may differ significantly in heavy smokers and non-smokers.

Noninvasive assays for oxidative RNA damage

Oxidative modification of human low-density lipoprotein (LDL) is thought to play an important role in the development of atherosclerosis. LDL oxidizability is believed to be strongly influenced by factors such as (a) content of preexisting lipid hydroperoxides (LOOHs) and (b) content of endogenous antioxidants such as α-tocopherol and β-carotene. The purpose of this study was to examine the prooxidant role of preexisting LDL-LOOHs, using a recently developed method for ultrasensitive and selective LOOH analysis: high-performance liquid chromatography with mercury drop electrochemical detection (HPLC-EC). Exceedingly low detection limits for LDL-LOOHs have been achieved by HPLC-EC, e.g., ∼100 fmol for cholesteryl ester hydroperoxide (CEOOH). This sensitivity has allowed us to monitor LDL-LOOHs at levels that are undetectable by most other methods. Fresh LDL prepared with the utmost care to prevent autoxidation was found to contain small, yet significant amounts of CEOOH, 6-12 pmol/mg protein. Our data suggest that these peroxides could not have arisen during LDL isolation or sample work-up for HPLC-EC. Incubation with GSH and phospholipid hydroperoxide glutathione peroxidase resulted in nearly complete reduction of the CEOOH. This LDL was found to be much more resistant to Cu2+-induced peroxidation than starting material, exhibiting a lag period that was at least six times greater. We have also determined that LDL becomes progressively more susceptible to Cu2+-induced lipid peroxidation (as evidenced by a shortened lag) when it is preloaded with increasing amounts of photochemically generated LOOHs. Taken together, these results provide strong support for the idea that preexisting LOOHs in LDL are important determinants of its overall oxidizability.

The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells.

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.

Probucol inhibits oxidative modification of low density lipoprotein.

Previous studies have established that low density lipoprotein (LDL) incubated with endothelial cells (EC) undergoes extensive oxidative modification in structure and that the modified LDL is specifically recognized by the acetyl LDL receptor of the macrophage. Thus, in principle, EC-modified LDL could contribute to foam cell formation during atherogenesis. Oxidatively modified LDL is also potentially toxic to EC. The present studies show that addition of probucol during the incubation of LDL with EC prevents the increase in the electrophoretic mobility, the increase in peroxides, and the increase in subsequent susceptibility to macrophage degradation. It has also been shown that oxidation of LDL catalyzed by cupric ion induces many of the same changes occurring during EC modification. Addition of probucol (5 microM) also prevented this copper-catalyzed modification of LDL. Most importantly, samples of LDL isolated from plasma of hypercholesterolemic patients under treatment with conventional dosages of probucol were shown to be highly resistant to oxidative modification either by incubation with endothelial cells or by cupric ion in the absence of cells. The findings suggest the hypothetical but intriguing possibility that probucol, in addition to its recognized effects on plasma LDL levels, may inhibit atherogenesis by limiting oxidative LDL modification and thus foam cell formation and/or EC injury. Other compounds with antioxidant properties might behave similarly.

The interaction of human plasma low density lipoproteins with glycosamino-glycans: influence of the chemical composition.

Human plasma of 5 normolipemic individuals was incubated for 24 hr at 37 C in the presence or in the absence of lecithin:cholesterol acyltransferase (LCAT)-inhibitors. Plasma stored at 4 C served as a control. The low density lipoprotein (LDL) fractions of the samples were isolated and investigated with respect to changes in chemical composition and complexing activity with glycosamino glycans (GAG). Incubation of plasma in the presence of LCAT inhibitors caused a significant increase of LDL triglycerides at the expense of cholesteryl esters. Incubation with active LCAT not only changed the core but also the surface constituents (decrease in phospholipids and in free cholesterol). The amount of GAG bound per mg of LDL was not uniformly changed in samples incubated after LCAT inhibition. LDL isolated from plasma incubated in the presence of LCAT, on the other hand, showed a significant reduction in GAG binding. The ratio of free cholesterol:GAG in the complex was most significantly reduced in LCAT-modified LDL. There was in addition a highly significant correlation between the LDL:GAG ratio in the complex and the free cholesterol and phospholipid content of the LDL samples. It is concluded that alterations in surface lipid constituents of LDL strongly affect their interaction with sulfated polysaccharides, an effect which may be relevant also in vivo for the interaction of LDL with cell surfaces and intercellular matrices.

Differential reactivity of human low density lipoproteins with monoclonal antibodies.

The immunoreactivities of LDL (low density lipoprotein) samples obtained from a variety of subjects were analyzed by comparing their capacities to compete with 125I-labeled LDL for binding to various monoclonal anti-LDL antibodies in competitive binding assays. A marked variation in epitope expression was observed. In comparison to an LDL standard, different preparations exhibited immunoreactivities (expressed as apparent apoB content) ranging from 30 to 400% of the LDL standard. Some epitopes were much more uniformly expressed than others. The number of epitopes expressed in different LDL preparations appeared to be related to the percentage composition of various lipid constituents in LDL. The results support the hypothesis that the epitope expression of apoB is modulated by the composition of the lipids associated with it.

A rapid and simple method for measurement of total protein in very low density lipoproteins by the Lowry assay

When total protein in very low density lipoprotein samples is measured by the method of Lowry et al. (1951. J. Biol. Chem 193:265-275), turbidity remains in the final color reaction. Addition of 0.1 ml of 2.5% (v/v) solution of Triton X-100 removes this turbidity immediately and effectively. This simple modification is faster, less cumbersome, and more economical than removing turbidity with ethyl ether or chloroform. Addition of 0.1 ml of 2.5% (w/v) sodium dodecyl sulfate to the final reaction mixture is also effective in removing turbidity and can be used as an alternative method.

Small-angle x-ray scattering and differential scanning calorimetry studies on reversibly modified human-serum low density lipoproteins

Small-angle x-ray scattering diagrams of human serum low density lipoprotein (LDL) were recorded at several temperatures in solutions of different freezing points. It was found that modifications of the x-ray patterns observed on cooling the lipoprotein samples below 0 degrees C are due to reversible alterations of the LDL surface structure induced by the freezing process (independent of temperature). With both intact and partially dehydrated LDL, differential scanning calorimetry (DSC) carried out in the body temperature range revealed a heat absorption characteristic of the transition from a liquid crystal to an isotropic liquid phase of cholesteryl esters within the lipoproteins (Deckelbaum, R. J., Shipley, R. J., Small, P. M., Lees, R. S., & George, P. K. (1975) Science 190, 392). However, small-angle x-ray scattering diagrams recorded with the same LDL sample before and after the partial removal of water were found to be very different: the scattering curve for intact LDL showed a strong band centered at (36 A)-1 which disappeared upon drying and reappeared upon restoring the water. Our results suggest that the presence of this signal strongly depends on the molecular structure of the lipoprotein surface.

The metabolism of low-density lipoprotein in a patient with familial hyperbetalipoproteinaemia.

1. Plasma low-density lipoprotein (LDL) from a patient with homozygous familial hyperbetalipoproteinaemia was labelled with 125I and LDL from a matched normal subject was labelled with 131I. The two samples of LDL were injected simultaneously into the patient and into a second normal subject. 2. In each subject the fractional rate of turnover of the two LDL samples was the same, but the fractional rate in the patient was less than half that in the normal subject. 3. It is concluded that the hyperbetalipoproteinaemia in the patient was not due to an abnormality in her LDL protein recognizable by her own body or by that of the normal recipient.

Charge heterogeneity of human low density lipoprotein (LDL).

Plasma samples were obtained from patients with known history of atherosclerosis as well as from a group of Bantus of South Africa and a group of medical students without any history of atherosclerotic vascular disease. The isoelectric point (IEP) of LDL of the different persons was determined by electrophoresis in agarose gel at different pH values. The IEP of the LDL of the students or of the Bantus, were fairly constant at pH 5.28; for the patients the IEP varied widely. The IEP of LDL from hypercholesteremic persons (Fredrickson type II) showed consistently a significantly higher IEP than for the controls. Electrophoretic mobility of the different samples of LDL was determined at pH 6.8 at different ionic strengths using NaCl or CaCl2. The effect of ionic strength (using NaCl) on the mobility was different for LDL isolated from different persons. Low concentrations of CaCl2 reverse the charge of lipoprotein from a negative to a positive one at pH 6.8. The drastic effect of Ca2+ on the net charge of LDL at pH 6.8, possibly due to the exposed phospholipid groups of LDL, shows that this ion may be important in the interaction between the lipoprotein and the mucopolysaccharides; such an interaction has been suggested by other workers as occurring in the arterial wall.