Low-density Lipoprotein Protein Research Articles

Pomegranate Wine Has Greater Protection Capacity Than Red Wine on Low-Density Lipoprotein Oxidation

Although there is a large body of evidence on the main role of red wine in protection of low-density lipoprotein (LDL) against oxidation, there are few data on the role of pomegranate juice, which has high phenolic content. We conducted this study considering the possible importance of pomegranate wine as an antioxidant and in order to make a comparison between red and pomegranate wines. The phenol levels of pomegranate and red wines (4,850 mg/L gallic acid equivalents and 815 mg/L gallic acid equivalents, respectively) were in accordance with their total antioxidant activity (39.5% and 33.7%, respectively). Both wines decreased LDL-diene levels following a 30-minute incubation period compared with controls (145 +/- 3.2 micromol/mg of LDL protein). However, pure pomegranate wine demonstrated a greater antioxidant effect (P < .01) on diene level (110 +/- 4.6 micromol/mg of LDL protein) than pure red wine (124 +/- 3.2 micromol/mg of LDL protein). In conclusion, we suggest that pomegranate wine has potential protective effects toward LDL oxidation, and it may be a dietary choice for people who prefer fruit wines.

No effect of menstrual cycle on LDL oxidizability and particle size

Objectives Premenopausal women have a lower incidence of cardiovascular disease than men, but this female advantage disappears after menopause, suggesting that female sex hormones exert some cardioprotective effects. One of the mechanisms proposed to explain this cardioprotection is the antioxidant properties of estrogens. The aim of this work was to assess whether fluctuations in ovarian hormones, particularly 17β-estradiol (E 2), during the menstrual cycle were associated with changes in the low-density lipoprotein (LDL) particle size, fatty acyl composition, α-tocopherol content and in vitro oxidizability. Methods Twenty-eight healthy premenopausal women (mean age: 32.2 years) participated in the study. Blood was drawn on days 3 (menstrual phase), 14 (follicular phase) and 22 (luteal phase) of the menstrual cycle for plasma determinations and LDL isolation. Plasma E 2, progesterone, follicle-stimulating hormone and luteinizing hormone were determined by immunoassay. LDL oxidation by Cu 2+- and 2,2′-azobis (2-amidinopropane) was measured by the formation of conjugated dienes, LDL particle size by quasi-elastic light scattering, fatty acyl composition by gas chromatography, α-tocopherol by reversed phase HPLC. A within-subjects analysis of variance was performed to determine significant differences of the variables over the course of a subject's menstrual cycle. Results The LDL oxidizability indices (lag time before the onset of propagation and the maximal oxidation rate) did not change during the menstrual cycle. The LDL particle size (24.8 ± 1.7 nm diameter), α-tocopherol (11.7 ± 3.7 nmol/mg LDL protein) and fatty acyl composition also remained constant. Conclusions The LDL physicochemical properties and oxidizability are not affected by menstrual cycle phase.

Exercise Attenuates Effects of Diabetic Dyslipidemia on the Low Density Lipoproteome

PURPOSE: We determined whether exercise training attenuates changes in the low density lipoprotein (LDL) proteome in diabetic dyslipidemia. Chronic endurance exercise has been demonstrated to reduce the incidence of coronary artery disease (CAD). However, the exact mechanism by which exercise exerts its cardioprotective effects is yet to be determined. A role for post-translationally modified (PTM) LDL has been proposed in the development of CAD. Diabetics are 3-4 times more likely to develop CAD and undergo enhanced LDL glycation, a PTM considered to accelerate atherogenesis. Interestingly, it has been documented that endurance athletes have lower levels of plasma LDL oxidation, one type of PTM. A decrease in the number and/or extent of LDL modifications may explain the cardioprotective effects of exercise. METHODS: Yucatan male pigs were low fat fed healthy control (C), high fat/cholesterol fed and alloxan-induced diabetic (diabetic dyslipidemic, DD), and DD that were aerobically exercise trained (DDX). Increased coronary vascular constriction in a swine model of diabetic dyslipidemia (DD) was prevented by exercise training (DDX) without decreasing total LDL concentration (Mokelke et al. J. Appl. Physiol. 95:1179, 2003). Thus, we possess a nearly ideal system in which we can test the hypothesis that changes in the number of modified proteins or the extent of modification is most closely associated with exercise. The lipoproteins, isolated by FPLC from normolipidemic controls, DD, and DDX pigs, were analyzed by 2-D gel electrophoresis and identified by peptide mass fingerprinting. RESULTS: Of 430 spots matched across all gel patterns analyzed, 171 spots were differentially expressed (p ≤ 0.05) and their identities determined. Of these, 22 changed with exercise (DDX vs. DD), of which only 2 decreased in abundance while the remaining 20 increased. Apolipoprotein E, an anti-atherogenic protein, demonstrated the greatest increase in abundance in the exercise trained group, DDX. To compare the extent of PTMs the charge modification index (CMI) of each LDL charge train was calculated. Numerous charge trains demonstrated increases in CMI in the DD, which likewise were attenuated with exercise training. One such charge train, phospholipase C, increased in overall abundance and CMI in DD and to a lesser extent in DDX, thus supporting our hypothesis. CONCLUSIONS: Similar effects in other LDL proteins suggest significant differences in LDL protein modification, the nature of which requires the use of tandem mass spectrometric techniques in a subsequent investigation. Supported by NIH RR13223, HL62552 (MS) and AFOSR 49620-002-1-0089 and FA9550-05-1-0216 (FW).

Defining Lipid-Interacting Domains in the N-Terminal Region of Apolipoprotein B

Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the "bad cholesterol" that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted alpha-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogeneous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC-dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous with lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins.

Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts

Previous studies have suggested that HDL retroendocytosis may play a role in scavenger receptor class B type I (SR-BI)-dependent selective lipid uptake in a cell-specific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and resecretion in fibroblast (COS-7) and hepatocyte (HepG2) cells expressing exogenous SR-BI. Approximately 17% and 24% of HDL associated in an SR-BI-dependent manner with COS-7 and HepG2 cells, respectively, accumulates intracellularly after a 10 min incubation. To determine whether this intracellular HDL undergoes retroendocytosis, we developed a pulse-chase assay whereby internalized biotinylated (125)I-HDL(3) secreted from cells is quantitatively precipitated from cell supernatants using immobilized streptavidin. Our results show a rapid secretion of a portion of intracellular HDL from both cell types (representing 4-7% of the total cell-associated HDL) that is almost complete within 30 min (half-life approximately 10 min). In COS-7 cells, the calculated rate of HDL secretion ( approximately 0.5 ng HDL/mg/min) was >30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake ( approximately 17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface ( approximately 19 ng HDL/mg/min) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cells was similar. BLT1, a compound that inhibits selective CE uptake, does not alter the amount of SR-BI-mediated HDL retroendocytosis in COS-7 cells. From these data, we conclude that HDL retroendocytosis in COS-7 and HepG2 cells is similar and that the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.

Protective effects of Peganum harmala L. extract, harmine and harmaline against human low-density lipoprotein oxidation

Oxidative modification of low-density lipoprotein (LDL) particles has been implicated in the process of atherogenesis. Antioxidants that prevent LDL from oxidation may reduce atherosclerosis. We have investigated the protective effect of Peganum harmala-extract (P-extract) and the two major alkaloids (harmine and harmaline) from the seeds of P. harmala against CuSO4-induced LDL oxidation. Through determination of the formation of malondialdehyde (MDA) and conjugated diene as well as the lag phase, the extract (P-extract) and compounds were found to possess an inhibitory effect. Moreover, harmaline and harmine reduced the rate of vitamin E disappearance and exhibited a significant free radical scavenging capacity (DPPH*). However, harmaline had a markedly higher antioxidant capacity than harmine in scavenging or preventive capacity against free radicals as well as inhibiting the aggregation of the LDL protein moiety (apolipoprotein B) induced by oxidation. The results suggested that P. harmala compounds could be a major source of compounds that inhibit LDL oxidative modification induced by copper.

Inflammation and Atherosclerosis

The simplistic view of atherosclerosis as a disorder of pathological lipid deposition has been redefined by the more complex concept of an ongoing inflammatory response. Apolipoprotein E and low-density lipoprotein (LDL)-receptor-deficient mice develop accelerated atherosclerosis allowing in-depth pathophysiological investigations. Atherosclerotic plaques in these mice contain large numbers of T cells and macrophages. Crossbreeding apolipoprotein E-deficient mice with T-cell-deficient mice and mice with impaired macrophage function (osteopetrotic op/op mice) disclosed the important impact of immune cells on atherosclerotic lesion development. In contrast to the detrimental role of T cells and macrophages, B cells appear to be atheroprotective. These basic experimental findings have partly been confirmed in studies of the human carotid artery system. Inflammation is not only instrumental in the development of human atheromatous plaques, but, importantly, plays a crucial role in the destabilization of internal carotid artery plaques, thus converting chronic atherosclerosis into an acute thrombo-embolic disorder. Humoral factors involved in internal carotid artery destabilization include cytokines, cyclooxygenase-2, matrix metalloproteinases, and tissue factor. Antibodies to oxidized LDL can reflect disease activity on one hand, but can also confer atheroprotection. Novel MRI techniques may aid in the in vivo assessment of acute plaque inflammation in humans. The impact of inflammation on the development of atherosclerotic plaques and their destabilization opens new avenues for treatment. The effects of statins, acetylsalicyclic acid and angiotensin-converting enzyme inhibitors on stroke prevention may partly be attributable to their profound anti-inflammatory actions. Vaccination against modified LDL and heat shock proteins halt plaque progression in experimental atherosclerosis. Their potential for prevention of human atherosclerosis is currently under investigation.

17β-Estradiol reverses shear-stress-mediated low density lipoprotein modifications

Within arterial bifurcations or branching points, oscillatory shear stress (OSS) induces oxidative stress mainly via the reduced nicotinamide adenine dinucleodtide phosphate (NADPH) oxidase system. It is unknown whether 17β-estradiol (E 2) can regulate OSS-mediated low-density lipoprotein (LDL) modifications. Bovine aortic endothelial cells were pretreated with E 2 at 5 nmol/L, followed by exposure to OSS (0 ± 3.0 dynes/cm 2 s and 60 cycles/min) in a flow system. E 2 decreased OSS-mediated NADPH oxidase mRNA expression, and E 2-mediated NO production was mitigated by the NO synthase inhibitor N(G)-nitro- l-argenine methyl ester. The rates of O 2 − production in response to OSS increased steadily as determined by superoxide-dismutase-inhibited ferricytochrome c reduction; whereas, pretreatment with E 2 decreased OSS-mediated O 2 − production ( n = 4, p < 0.05). In the presence of native LDL (50 μg/mL), E 2 also significantly reversed OSS-mediated LDL oxidation as determined by high-performance liquid chromatography. In the presence of O 2 − donor, xanthine oxidase (XO), E 2 further reversed XO-induced LDL lipid peroxidation ( n = 3, p < 0.001). Mass spectra acquired in the m/ z 400–1800 range, revealed XO-mediated LDL protein nitration involving tyrosine 2535 in the α-2 domains, whereas pretreatment with E 2 reversed nitration, as supported by the changes in nitrotyrosine intensities. Thus, E 2 plays an indirect antioxidative role. In addition to upregulation of endothelial NO synthase and downregulation of Nox4 expression, E 2 influences LDL modifications via lipid peroxidation and protein nitration.

A Tale of Two Trials

The Aggrastat to Zocor (A to Z) and Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE IT) trials compared intensive and moderate statin therapy after acute coronary syndromes, with seemingly disparate results. We analyzed the design, implementation, and results of the two trials in an attempt to clarify the effects of early intensive statin therapy. Study design, end points, and definitions were compared. In each trial, comparisons were made between intensive and moderate arms for both trials' primary end points and death/myocardial infarction. Analyses were performed over various time points: at the end of the trials, < or =4 months, and >4 months. Subjects in A to Z had higher-risk demographics. More PROVE IT subjects were enrolled in the United States and underwent prerandomization revascularization. The low-density lipoprotein (LDL) difference was greater in A to Z than in PROVE IT early (< or =4 months) but less late. Significant C-reactive protein reduction was earlier in PROVE IT. With common end points, event rates were higher in A to Z, and early favorable separation of event curves was seen in PROVE IT but not in A to Z. Clinical end point rates and reductions were similar in both trials after 4 months. An early benefit was seen in PROVE IT but not in A to Z. Late-phase results were similar. Factors that may explain this disparity include the intensity of therapy in the early phase, timing, and magnitude of LDL and C-reactive protein lowering, differences in early revascularization, and the play of chance. Taken together, the results of these trials support a strategy of early intensive statin therapy coupled with revascularization when appropriate in patients after acute coronary syndrome.

Pharmacological characterization in vitro of EP2306 and EP2302, potent inhibitors of squalene synthase and lipid biosynthesis

We investigated the effects of EP2306 and EP2302, two novel 2-biphenylmorpholine derivatives, on squalene synthase activity in rabbit and human liver microsomes, lipid biosynthesis, low-density lipoprotein (LDL) receptor expression and LDL protein uptake as well as apoB secretion in HepG2 cells. Both EP2306 and EP2302 inhibited squalene synthase activity dose-dependently. In rabbit liver microsomes, the IC 50 values were 33 μM for EP2306 and 0.6 μM for EP2302 whereas in human liver microsomes, they were 63 μM for EP2306 and 1 μM for EP2302. Both EP2300 compounds inhibited cholesterol production by HepG2 cells dose dependently with IC 50 values of 13.3 μM for EP2306 and 3 μM for EP2302. Furthermore, both EP2300 compounds and simvastatin significantly reduced triglyceride synthesis and apoB secretion and increased LDL receptor expression and LDL uptake in HepG2 cells. In summary, we have shown that EP2300 compounds are potent inhibitors of squalene synthase activity in rabbit and human liver microsomes and also they are effective inhibitors of cholesterol and triglyceride biosynthesis in HepG2 cells. These results suggest that EP2306 and EP2302 might prove to be useful for lipid-lowering and treatment of atherosclerosis in vivo.

Mechanisms of Dysregulation of Low-Density Lipoprotein Receptor Expression in Vascular Smooth Muscle Cells by Inflammatory Cytokines

Although inflammation is a recognized feature of atherosclerosis, the impact of inflammation on cellular cholesterol homeostasis is unclear. This study focuses on the molecular mechanisms by which inflammatory cytokines disrupt low-density lipoprotein (LDL) receptor regulation. IL-1beta enhanced transformation of vascular smooth muscle cells into foam cells by increasing uptake of unmodified LDL via LDL receptors and by enhancing cholesterol esterification as demonstrated by Oil Red O staining and direct assay of intracellular cholesterol concentrations. In the absence of IL-1beta, a high concentration of LDL decreased LDL receptor promoter activity, mRNA synthesis and protein expression. However, IL-1beta enhanced LDL receptor expression, overriding the suppression usually induced by a high concentration of LDL and inappropriately increasing LDL uptake. Exposure to IL-1beta also caused overexpression of the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP), and enhanced its translocation from the endoplasmic reticulum to the Golgi, where it is known to cleave SREBP, thereby enhancing LDL receptor gene expression. These observations demonstrate that IL-1beta disrupts cholesterol-mediated LDL receptor feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam cell formation. The implication of these findings is that inflammatory cytokines may contribute to intracellular LDL accumulation without previous modification of the lipoprotein.

Persistent High Levels of Plasma Oxidized Low-Density Lipoprotein After Acute Myocardial Infarction Predict Stent Restenosis

Recently, elevated levels of plasma oxidized low-density lipoprotein (LDL) have been shown to relate to plaque instability in human atherosclerotic lesions. We investigated prospectively patients admitted with acute myocardial infarction (AMI) who underwent primary coronary stenting to evaluate whether the 6-month outcome could be predicted by measuring plasma oxidized LDL (ox-LDL) levels at the time of hospital discharge. Plasma ox-LDL levels were measured in 102 patients with AMI undergoing primary coronary stenting using a highly sensitive ELISA method. Measurements were taken on admission and at discharge, and the findings related to the clinical outcome. At 6-month follow-up, angiographic stent restenosis occurred in 25 (25%) of the 102 AMI patients. Plasma ox-LDL levels at discharge were significantly (P=0.0074) higher in the restenosis group than those in the no-restenosis group (1.03+/-0.65 versus 0.61+/-0.34 ng/5 microg LDL protein). Multiple regression analysis showed that only plasma ox-LDL levels at discharge were a statistically significant independent predictor for late lumen loss after stenting (beta=0.645; P<0.0001). This prospective study demonstrates that persistence of an increased level of plasma ox-LDL at discharge is a strong independent predictor of stent restenosis at 6-month follow-up in AMI patients.

Decrease in pH Strongly Enhances Binding of Native, Proteolyzed, Lipolyzed, and Oxidized Low Density Lipoprotein Particles to Human Aortic Proteoglycans

Binding of low density lipoprotein (LDL) to proteoglycans and modification of LDL are key processes in atherogenesis. Recently, it has been demonstrated that during atherogenesis the extracellular pH of atherosclerotic lesions decreases. We have examined the effect of the decreased pH on the binding of LDL to human aortic proteoglycans. The binding of native, oxidized, proteolyzed (alpha-chymotrypsin-treated), or lipolyzed (sphingomyelinase- or phospholipase A(2)-treated) LDL particles to proteoglycans were measured in microtiter well assays at pH 5.5-7.5. We found that the lower the pH, the higher the amount of binding of LDL to proteoglycans. At the lowest pH tested (pH 5.5), the amounts of proteoglycan-bound native, proteolyzed, sphingomyelinase-, and phospholipase A(2)-treated LDL were 20-, 23-, 30-, and 37-fold higher, respectively, than at pH 7.5. Interestingly, although oxidized LDL failed to bind to proteoglycans at neutral pH, there was significant binding at acidic pH. Binding of native and modified LDL to proteoglycans at pH 5.5 was blocked by 1 m NaCl, indicating that at neutral pH LDL binds to proteoglycans via ionic interactions. Inhibition of this binding by acetylation and cyclohexanedione treatment of LDL showed that the positively charged amino acids of apolipoprotein B-100, lysine, and arginine, respectively, mediated the ionic interaction. Taken together, our results suggest that in areas of atherosclerotic arterial intima where the extracellular pH decreases, retention of LDL by proteoglycans is enhanced, leading to extracellular accumulation of LDL and progression of the disease.

Selective uptake and efflux of cholesteryl linoleate in LDL by macrophages expressing 12/15-lipoxygenase

Oxidation of low density lipoprotein (LDL) is a critical step for atherogenesis, and the role of the 12/15-lipoxygenase (12/15-LOX) as well as LDL receptor-related protein (LRP) expressed in macrophages in this process has been suggested. The oxygenation of cholesteryl linoleate in LDL by mouse macrophage-like J774A.1 cells overexpressing 12/15-LOX was inhibited by an anti-LRP antibody but not by an anti-LDL receptor antibody. When the cells were incubated with LDL double-labeled by [ 3H]cholesteryl linoleate and [ 125I]apoB, association with the cells of [ 3H]cholesteryl linoleate expressed as LDL protein equivalent exceeded that of [ 125I]apoB, indicating selective uptake of [ 3H]cholesteryl linoleate from LDL to these cells. An anti-LRP antibody inhibited the selective uptake of [ 3H]cholesteryl ester by 62% and 81% with the 12/15-LOX-expressing cells and macrophages, respectively. Furthermore, addition of LDL to the culture medium of the [ 3H]cholesteryl linoleate-labeled 12/15-LOX-expressing cells increased the release of [ 3H]cholesteryl linoleate to the medium in LDL concentration- and time-dependent manners. The transport of [ 3H]cholesteryl linoleate from the cells to LDL was also inhibited by an anti-LRP antibody by 75%. These results strongly suggest that LRP contributes to the LDL oxidation by 12/15-LOX in macrophages by selective uptake and efflux of cholesteryl ester in the LDL particle.

Effect of bicarbonate on iron-mediated oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) may play an important role in atherosclerosis. We studied the effects of bicarbonate/CO2 and phosphate buffer systems on metal ion-catalyzed oxidation of LDL to malondialdehyde (MDA) and to protein carbonyl and MetO derivatives. Our results revealed that LDL oxidation in mixtures containing free iron or heme derivatives was much greater in bicarbonate/CO2 compared with phosphate buffer. However, when copper was substituted for iron in these mixtures, the rate of LDL oxidation in both buffers was similar. Iron-catalyzed oxidation of LDL was highly sensitive to inhibition by phosphate. Presence of 0.3-0.5 mM phosphate, characteristic of human serum, led to 30-40% inhibition of LDL oxidation in bicarbonate/CO2 buffer. Iron-catalyzed oxidation of LDL to MDA in phosphate buffer was inhibited by increasing concentrations of albumin (10-200 microM), whereas MDA formation in bicarbonate/CO2 buffer was stimulated by 10-50 microM albumin but inhibited by higher concentrations. However, albumin stimulated the oxidation of LDL proteins to carbonyl derivatives at all concentrations examined in both buffers. Conversion of LDL to MDA in bicarbonate/CO2 buffer was greatly stimulated by ADP, ATP, and EDTA but only when EDTA was added at a concentration equal to that of iron. At higher than stoichiometric concentrations, EDTA prevented oxidation of LDL. Results of these studies suggest that interactions between bicarbonate and iron or heme derivatives leads to complexes with redox potentials that favor the generation of reactive oxygen species and/or to the generation of highly reactive CO2 anion or bicarbonate radical that facilitates LDL oxidation.

Catabolism of native and oxidized low density lipoproteins: in vivo insights from small animal positron emission tomography studies

The human organism is exposed to numerous processes that generate reactive oxygen species (ROS). ROS may directly or indirectly cause oxidative modification and damage of proteins. Protein oxidation is regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to Alzheimer's disease and atherosclerosis. As a representative example, oxidation of low density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development and outcome of diseases are scarce. One reason for this is the shortage of methods for direct assessment of the metabolic fate of circulating oxLDL in vivo. We present an improved methodology based on the radiolabelling of apoB-100 of native LDL (nLDL) and oxLDL, respectively, with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Radiolabelling of both nLDL and oxLDL using [(18)F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively, in vitro. The method was further evaluated with respect to the radiopharmacological properties of both [(18)F]fluorobenzoylated nLDL and oxLDL by biodistribution studies in male Wistar rats. The metabolic fate of [(18)F]fluorobenzoylated nLDL and oxLDL in rats in vivo was further delineated by dynamic positron emission tomography (PET) using a dedicated small animal tomograph (spatial resolution of 2 mm). From this study we conclude that the use of [(18)F]FB-labelled LDL particles is an attractive alternative to, e.g., LDL iodination methods, and is of value to characterize and to discriminate the kinetics and the metabolic fate of nLDL and oxLDL in small animals in vivo.

Antioxidant Activities of Phenolic Acids on Ultraviolet Radiation-Induced Erythrocyte and Low Density Lipoprotein Oxidation

The exposure of mammalian cells to UV light induces various deleterious responses. Some of the major harmful effects are DNA damage, cell membrane peroxidation, systemic immune suppression, and aging acceleration. Reactive oxygen species and free radicals are believed to be largely responsible for some of the deleterious effects of UV upon cells. Typical administration of antioxidants has recently proved to represent a successful strategy for protecting the cells against UV-mediated oxidative damage. The objective of this study was to investigate the inhibitory effect of phenolic acids (caffeic acid, ferulic acid, gallic acid, and protocatechuic acid) on oxidative damage in human erythrocytes and low-density lipoprotein (LDL) induced by UVB radiation. The results revealed that the thiobarbituric acids reactive substances induced by UVB were decreased from 2.78 to 0.12-0.89 nmol MDA/mg protein in erythrocyte ghost and from 0.72 to 0.14-0.43 nmol MDA/mg protein in LDL by the addition of phenolic acids (100 muM). Caffeic acid, ferulic acid, and gallic acid exhibited over 85 and 60% inhibitory effect toward UVB-induced oxidation in erythrocytes and LDL, respectively. Phenolic acids, especially gallic acid, could maintain the normal glutathione levels and glutathione peroxidase activity in hemolysate from erythrocytes that were exposed to UVB radiation in comparison with untreated control. The results indicate that the antioxidant activities of caffeic acid and ferulic acid play a potential role in protection against UVB oxidative damage to human erythrocytes and LDL.

Caponization and testosterone implantation effects on blood lipid and lipoprotein profile in male chickens

To understand the role of lipid metabolism in increasing body fat accumulation after caponization of male chickens, trials were conducted to determine the effects of levels of testosterone implantation on lipoprotein composition. Male chickens were caponized at 12 wk and selected at 16 wk for a 10-wk feeding experiment. Fifteen male and 15 caponized (capon) chickens were used in trial 1. Ten sham operated chickens (sham) and 40 capons were randomly divided among 4 treatments in trial 2; the treatments were as follows: implantation of cholesterol (1.62 mm i.d. x 3.16 mm o.d., 9.24+/-0.36 mg) or implantation of testosterone at low (1 mm i.d. x 3 mm, o.d., 5.88+/-0.23 mg), medium (1.62 mm i.d. x 3.16 mm, o.d., 9.81+/-0.17 mg), or high (2 mm i.d. x 4 mm, o.d., 16.7+/-0.24 mg) dose. The results of trial 1 showed that caponization decreased (P < 0.05) blood testosterone concentrations and increased (P < 0.05) abdominal fat weight and relative abdominal fat weight in capons. Caponization also increased low density lipoprotein (LDL), high density lipoprotein (HDL), LDL protein, and HDL protein and decreased LDL-free cholesterol (LDL-FC), HDL-FC, and HDL-phospholipid (HDL-PL) percentages (P < 0.05). In trial 2 capons implanted with increasing testosterone levels exhibited proportional increases in blood testosterone concentration, although blood testosterone concentration in implanted capons were not fully restored to those of the sham group. High dose testosterone implantation inhibited abdominal fat accumulation and increased glucose and glycerol concentrations compared with the cholesterol implantation. Caponization of male chickens decreased the androgen level and increased the blood triacylglyceride content. Caponization also changed the lipoprotein profiles, which resulted in increased lipid storage capacity. The testosterone concentration, therefore, must achieve threshold concentrations to inhibit lipid accumulation in the testosterone implanted capon.

Low density lipoprotein non-esterified fatty acids and lipoprotein lipase in diabetes

Objectives: Fatty acid metabolism is disturbed in poorly controlled diabetes. Low density lipoprotein (LDL) oxidation, thought to be an atherogenic modification, is partly dependent on LDL fatty acid content whether it be in the form of cholesteryl ester, phospholipids, triglyceride or non-esterified fatty acid (NEFA). Lipoprotein lipase (LPL) is deficient in diabetic patients. Lipoprotein lipase bound to LDL may facilitate cholesterol accumulation in the artery wall through the attachment of LDL to the proteoglycans expressed on endothelial cells and collagen. The purpose of this study was to examine the degree of binding of fatty acids and lipoprotein lipase to LDL in type 2 diabetic patients and to examine the relationship between non-esterified fatty acids attached to LDL and LDL oxidisability. Subjects and methods: Eight type 2 diabetic patients and eight control subjects were examined fasting and at 4 and 6 h following a high fat meal. Six control subjects were examined fasting and 30 min after intravenous heparin. LDL was isolated by sequential ultracentrifugation. Individual LDL non-esterified fatty acids were measured by gas–liquid chromatography following transmethylation. LPL and oxidised LDL were measured by ELISA. Results: The diabetic patients had HbA1c of 7.8 ± 0.5% confirming moderate diabetic control. There was a large increase in the mean non-esterified fatty acids on LDL from diabetic subjects (0.66 ± 0.40 mg/mg versus 0.06 ± 0.02 mg/mg LDL protein, p < 0.01). Mean LDL cholesterol ester fatty acids were also significantly increased in the diabetic subjects (1.47 ± 0.58 mg/mg versus 0.57 ± 0.40 mg/mg LDL protein, p < 0.01). There was a significant increase in oxidised LDL (31.2 ± 24 mg/mg versus 7.7 ± 4.5 mg/mg LDL protein, p < 0.01) and a significant correlation between postprandial non-esterified fatty acid and LDL oxidation ( r = 0.69, p < 0.05). LPL was significantly increased on the LDL but not in the plasma of diabetic subjects. Acute elevation in non-esterified fatty acids produced by heparin in control subjects did not increase LDL non-esterified fatty acids. Conclusions: This study demonstrates that the disturbance in fatty acid metabolism found in type 2 diabetic subjects is associated with a significant increase in non-esterified fatty acids attached to LDL. This may account, at least in part, for the increased oxidation of the LDL and therefore its atherogenicity. The finding of an increase in the amount of LPL bound to LDL suggests an important mechanism to facilitate the uptake of diabetic LDL by endothelial proteoglycans and collagen in the atherosclerotic plaque.

Lipoproteomics I: Mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.