Low Density Lipoprotein Apoprotein Research Articles

Dextran sulfate bulk and surface-modified microfiltration membrane for simultaneous blood plasma harvesting and low-density lipoprotein removal during plasmapheresis

Plasmapheresis is currently used for the harvesting of blood plasma in medical labs and plasma donation centers. This is a fast, single-step membrane process for both collection of blood plasma and simultaneous blood cell isolation. This paper presents the development of a novel membrane that can be used to simultaneously harvest blood plasma and isolate low-density lipoprotein (LDL), known as bad cholesterol, from the blood. To achieve this goal, dual surface and bulk modification by dextran sulfate (DS) was performed on a polyethersulfone (PES) membrane containing single wall carbon nanotubes (SWCNT). DS is an anionic ligand that repels negatively charged substances like red blood cells (RBCs) and adsorbs positively charged substances like Apo B100; the apoprotein of LDL. Scanning Electron Microscope (SEM), Energy Dispersive X-Ray (EDX), and Fourier Transform Infrared (FTIR) spectroscopy analyses provided evidence of the grafting of DS on single wall carbon nanotubes (DS-SWCNT). Surface studies including water contact angle, zeta potential analysis, surface morphology, Attenuated Total Reflectance-FTIR (ATR-FTIR), and EDX, confirmed the presence of DS on the PES membrane surface. The SEM cross-section results showed the formation of a two-zone morphology with an open structure on the upside and a dense structure on the downside that provide optimal conditions for efficient separation of blood cells. The complete blood count (CBC) and biochemistry analyses revealed dual bulk and surface-modified membrane, and a specific sample (DM2) showed the best performance for blood cells and LDL separation. The maximum separation of blood cells was 87% RBC, 98% white blood cells (WBCs), and 83% platelets (PLTs). Moreover, DM2 exhibited a sieving ratio for LDL of 0.33. Blood compatibility analyses confirmed that DM2 and other related samples had low hemolysis and prolonged coagulation times.

Interaction of different lipoprotein types with cholesterol at the air/water interface

Cholesterol (Chol) interacts with lipoproteins, in order to be transported through the aqueous bloodstream. High density lipoproteins (HDL) and low density lipoproteins (LDL) transport cholesterol differently, a result that may be due to a difference in their interactions with cholesterol. Here, we investigated how the lipoprotein type affects the interaction with cholesterol by using a Langmuir trough and fluorescence microscope. We studied pure monolayers of 1) Chol, 2) LDL, and 3) HDL, and mixed monolayers of 1) Chol-LDL, and 2) Chol-HDL at air/water interfaces. Images of the Chol-LDL mixed monolayer showed many small sterol domains distributed in the non-sterol molecules (e.g. phospholids, proteins and lipids) of LDL. The sterol domains that were seen in the Chol-HDL mixed monolayer were larger in size but smaller in number than those seen in the Chol-LDL mixed monolayers. These images and the excess area, excess free energy, and free energy of mixing values obtained from the thermodynamic analysis of the surface pressure-area per molecule isotherms suggested that the cholesterol phase separated more from HDL than from LDL. Cholesterol was therefore concluded to interact with LDL better than with HDL. This more favorable interaction was explained by the presence of hydrophobic interactions between cholesterol and Apo-B, the major apoprotein of LDL.

My Early Days with Ari Helenius: Detergents and Viruses.

My Early Days with Ari Helenius: Detergents and Viruses.

Phytosterols, Phytostanols, and Lipoprotein Metabolism

The efficacy of phytosterols and phytostanols added to foods and food supplements to obtain significant non-pharmacologic serum and low density lipoprotein (LDL) cholesterol reduction is well documented. Irrespective of age, gender, ethnic background, body weight, background diet, or the cause of hypercholesterolemia and, even added to statin treatment, phytosterols and phytostanols at 2 g/day significantly lower LDL cholesterol concentration by 8%–10%. They do not affect the concentrations of high density lipoprotein cholesterol, lipoprotein (a) or serum proprotein convertase subtilisin/kexin type 9. In some studies, phytosterols and phytostanols have modestly reduced serum triglyceride levels especially in subjects with slightly increased baseline concentrations. Phytosterols and phytostanols lower LDL cholesterol by displacing cholesterol from mixed micelles in the small intestine so that cholesterol absorption is partially inhibited. Cholesterol absorption and synthesis have been carefully evaluated during phytosterol and phytostanol supplementation. However, only a few lipoprotein kinetic studies have been performed, and they revealed that LDL apoprotein B-100 transport rate was reduced. LDL particle size was unchanged, but small dense LDL cholesterol concentration was reduced. In subjects with metabolic syndrome and moderate hypertriglyceridemia, phytostanols reduced not only non- high density lipoprotein (HDL) cholesterol concentration but also serum triglycerides by 27%, and reduced the large and medium size very low density lipoprotein particle concentrations. In the few postprandial studies, the postprandial lipoproteins were reduced, but detailed studies with apoprotein B-48 are lacking. In conclusion, more kinetic studies are required to obtain a more complete understanding of the fasting and postprandial lipoprotein metabolism caused by phytosterols and phytostanols. It seems obvious, however, that the most atherogenic lipoprotein particles will be diminished.

Misfolding of Apoprotein B-100, LDL Aggregation and 17-β -estradiol in Atherogenesis

The long quest for a missing mechanistic rationale accounting for the correlation between plasma cholesterol levels and cardiovascular disease (CVD) has been focused on various possible modifications of low density lipoprotein (LDL), turning this physiological cholesterol carrier into a damaging agent able to trigger atherogenesis and later the onset of the disease. In addition to the debated oxidized LDL (oxLDL), a modified LDL with a misfolded apoprotein B-100, called electronegative LDL(-) for its negative charge due to an increased amount of free fatty acids, is commonly present in plasma. LDL(-) is generated by the action of secretory calcium dependent phospholipase A2. LDL(-) primes LDL aggregation and amyloid formation according to mechanisms very similar to those observed in other misfolding diseases. The LDL particle aggregates recall the structure and size of the subendothelial lipid droplets described in early atherogenesis and elicit a powerful inflammatory response. The use of 17-β-estradiol (E2) confirmed that the suggested atherogenicity of LDL (-) is mostly dependent on the misfolded character of its apoprotein. E2 binding to the apoprotein of native LDL, through a specific and saturable receptor, inhibits misfolding phenomenon despite an unaffected production of LDL (-) by phospholipase A2, ultimately preventing LDL aggregation. The apoprotein misfolding in LDL(-) emerges as a possible significant trigger mechanism of atherogenesis. Potential implications for the development of novel therapeutic approaches might be hypothesized in perspective. The existing evidence is discussed and reported in this review.

Dyslipidemia in chronic kidney disease: Causes and consequences

Cardiovascular risk increases with each decrement in renal function. Abnormalities in plasma lipoprotein composition among patients with end-stage renal disease represent a change both in lipoprotein level and structure. Low-density lipoprotein (LDL) cholesterol is not increased and LDL level is not strongly correlated with outcome. In contrast, high-density lipoprotein (HDL) levels are decreased and are inversely associated with cardiovascular risk. HDL is decreased because of an increased rate of catabolism associated with a predominance of small dense HDL. HDL fails to mature normally as a result of decreased lecithin cholesterol ester (CE) transfer protein (lecithin:cholesterol acyltransferase), leaving CE poor, triglyceride (TG) rich HDL3, and pre β HDL. Chronic kidney disease (CKD) also causes insulin resistance, and this may also contribute to low HDL levels. Plasma TG levels are increased and TGs are found in an expanded intermediate density lipoprotein (IDL) pool. These are composed of chylomicron and very low-density lipoprotein remnants resulting from delayed lipolysis on the endothelium and impaired hepatic uptake. IDL in contrast to LDL is associated with vascular disease in dialysis patients. Lipoprotein (a) (Lp (a)) levels are also increased. In patients without renal disease. The concentration of Lp (a) is inversely associated with the size of the apolipoprotein (a) isoform inherited; Lp (a) levels are increased in patients with kidney disease as a consequence of increased concentrations, primarily of the high molecular weight isoform. Lp (a) levels are also associated with cardiovascular outcome among dialysis patients.

Distribution of low‐density lipoprotein‐bound low‐molecular‐weight thiols: A new analytical approach

We have recently demonstrated that low-density lipoprotein (LDL) apoprotein is able to bind the most concentrated plasma thiols such as cysteine, cysteinylglycine, and homocysteine by disulfide linkage. However, the LIF CE assay employed to measure linked thiols was not sensitive enough to verify whether low concentrated plasma thiols as glutathione and glutamylcysteine are also linked to apoprotein. By modifying sample treatment and electrophoretic parameters we set up a new method with an LOQ of about 1.5 nmol/L, by which we demonstrate that LDL apoprotein binds all physiological plasma thiols. The increased sensitivity was obtained by drying released apoB thiols after reduction treatment, dissolving them directly in a low volume of derivatization buffer and decreasing the dilution factor of derivatized sample before CE injection. Moreover, by increasing the concentration of the electrolyte buffer, we improved the selectivity of peaks, in particular between glutathione (GSH) and the impurity peak derived from unreacted 5-iodoacetamidofluorescein, which in the previous electrophoretic conditions were overlapped. The method optimization, reached by searching the best combination between sample matrix and CE run buffer, is fully described. Given the potential pathologic significance of protein thiolation, the proposed method may be useful to understand the mechanisms and the balances that regulate the interaction between thiols and -SH free groups of proteins.

Dietary and Genetic Probes of Atherogenic Dyslipidemia

A goal of dietary management of cardiovascular disease risk in patients with obesity and metabolic syndrome is improvement in the atherogenic dyslipidemia comprising elevated triglyceride, reduced high-density lipoprotein (HDL) cholesterol, and increased numbers of small, dense low-density lipoprotein (LDL) particles. Individuals with a genetically influenced trait characterized by a high proportion of small, dense LDL (phenotype B) respond to a low-fat, high-carbohydrate diet with greater reduction of LDL cholesterol, apoprotein B, and mid-sized LDL2 particles than unaffected subjects (phenotype A). In contrast, in phenotype A subjects there is a reciprocal shift from large LDL1 to small LDL3 such that a high proportion convert to phenotype B. There is evidence for heritable effects on these diet-induced subclass changes and for the involvement of specific genes. For example, a haplotype of the APOA5 gene associated with increased plasma triglyceride and small, dense LDL predicts greater diet-induced reduction of LDL2, a haplotype-specific effect that is strongly correlated with both increased VLDL precursors and LDL4 products. Understanding of such diet-genotype interactions may help to elucidate mechanisms that are responsible for phenotype B and for its differential dietary responsiveness. This information may also ultimately help in identifying those individuals who are most likely to achieve cardiovascular risk benefit from specific dietary interventions.

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: Implication in hemodialysis mediated atherogenic LDL modification

Objective: Al3+ stimulates Fe2+ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al3+ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al3+ on LDL oxidation.Materials and methods: Using different LDL modifying systems (Fe2+, Cu2+, free radical generating compounds, human endothelial cells, hemin/H2O2 and HOCl), the influence of Al3+ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.Results: Al3+ could stimulate the oxidizability of LDL by Fe2+, but not in the other systems tested. Al3+ and Fe2+ were found to bind to LDL and Al3+could compete with Fe2+ binding to the lipoprotein. Fluorescence polarization data indicated that Al3+ does not affect the phospholipid compartment of LDL.Conclusions:The results indicate that increased LDL oxidation by Fe2+ in presence of Al3+ might be due to blockage of Fe2+ binding sites on LDL making more free Fe2+ available for lipid oxidation.

Hypochlorous acid scavenging properties of local mediterranean plant foods

Oxidative modification of low density lipoprotein (LDL) is involved in the pathogenesis of atherosclerosis and coronary heart disease, which are low in the Mediterranean area possibly due to a high dietary proportion of plant foods. Ethanolic extracts were prepared from more than 120 Mediterranean edible plants collected in remote areas (which maintain their traditional diet) and their antioxidant potential was studied. Extracts derived from Agaricus campestris, Cynara cardunculus, Thymus pulegioides, and Vicia faba were subjected to further analysis in this study. The extracts' potential to scavenge the DPPH radical (2,2-diphenyl-1-picrylhydrazyl radical) and hypochlorous acid (HOCl), as well as their antioxidant capacity, was comparable to the those obtained for standard antioxidants (e.g., quercetin, Trolox). Myeloperoxidase (MPO) catalyzes the production of the highly chlorinating and oxidizing agent HOCl, which reacts with the LDL apoprotein moiety, leading to the derivatization of its aminoacidic residues. Coincubation with extracts significantly prevented HOCl-induced modification of the LDL residue tryptophan, whereas higher concentrations were required to retard lysine damage. Moreover, the extracts inhibited MPO-catalyzed guaiacol oxidation in a concentration-dependent manner in a cell-free assay but, in contrast, did not affect MPO activity in isolated human neutrophils. MPO is also known to facilitate nitric dioxide oxidation. The formation of 3-nitrotyrosine was significantly lower in bovine endothelial aortic cells incubated with C. cardunculus or T. pulegioides. In synthesis, our study shows that local Mediterranean plant foods prevent HOCl toxicity in vitro and, thus, suggests further mechanisms responsible for the reported health-beneficial effect of the Mediterranean diet.

Determination of the estrogenic and antiestrogenic effects of environmental contaminants in chicken embryo hepatocyte cultures by quantitative-polymerase chain reaction.

A method was developed to measure the estrogenic and antiestrogenic effects of various chemicals and organochlorine extracts in chicken embryo primary hepatocyte cultures. Messenger RNAs (mRNAs) for the estrogen-inducible egg yolk proteins, vitellogenin II (VTGII), and very low-density lipoprotein apoprotein II (apoII), were measured by multiplex quantitative reverse transcription-polymerase chain reaction (Q-PCR). After 48 h of exposure, both VTGII and apoII mRNA levels were induced by moxestrol (1-1,000 nM), 17 beta-estradiol (10-1,000 nM), o,p'-DDT (apoII: 1,000 and 10,000 nM, VTGII: 10,000 nM), 4-tertoctylphenol ([OP]; apoII: 20 and 50 microM, VTGII: 10-50 microM), and methoxychlor ([MXCL]; apoII: 5-50 microM, VTGII: 20 and 50 microM). Tamoxifen (100 and 1,000 nM) induced apoII mRNA only, and bisphenol A (BPA) was not estrogenic. Inhibition of moxestrol-mediated VTGII or apoII mRNA induction by MXCL, o,p'-DDT and tamoxifen indicated that these chemicals were also antiestrogenic at concentrations similar to those which caused estrogenic responses. Organochlorine extracts prepared from herring gull embryo yolk sacs obtained from three Great Lakes sites and one Atlantic coast site (reference site) did not show any estrogenic activities. However, the same extracts from all three Great Lakes sites had antiestrogenic activities. These results indicate that wild birds may be susceptible to the estrogenic or antiestrogenic effects of environmental contaminants.

Very low-density lipoprotein-apoprotein CI is increased in diabetic nephropathy: Comparison with apoprotein CIII

Recent studies have suggested that apoprotein (apo) CI in very low-density lipoprotein (VLDL) plays an important role in causing hypertriglyceridemia independent of apo CIII, which is associated with coronary heart disease (CHD). Because the incidence of CHD is increased in diabetic patients and is even higher when diabetic nephropathy is developed, we measured apo CI levels in VLDL from type 2 diabetic patients, with various degree of nephropathy, and compared the results with those for healthy controls or nondiabetic patients with chronic renal failure (CRF). This study enrolled healthy control subjects, type 2 diabetic patients with normoalbuminuria, microalbuminuria, overt proteinuria, and CRF on hemodialysis and nondiabetic hemodialysis patients. VLDL (density <1.006) was separated by ultracentrifugation. Then the apo CI, CIII, and B concentrations in VLDL were measured by enzyme-linked immunosorbent assay (ELISA). The apo CI, CIII, and B concentrations in VLDL were respectively 3-, 2-, and 2-fold higher, respectively, in diabetic patients with overt proteinuria than in controls. Hemodialysis patients with diabetic nephropathy had levels of apo CI, CIII, and B in VLDL that were 2.6-, 2.7- and 2-fold higher, respectively, than those in controls. Nondiabetic hemodialysis patients also had a 2.7-fold higher level of VLDL apo CIII, whereas VLDL apo CI and VLDL apo B were not significantly increased. VLDL apo CI was significantly correlated with VLDL apo B independently of VLDL apo CIII level. An increase of VLDL apo CIII is a prominent feature of dyslipidemia in CRF patients, regardless of whether they are diabetic or nondiabetic, whereas an increase of VLDL apo CI is more specific to diabetic nephropathy and is closely associated with an increase of VLDL particle numbers, a new risk factor for CHD.

The effect of 7-year infancy-onset dietary intervention on serum LDL and HDL cholesterol subclasses in healthy children: The prospective, randomised strip study

The oxidation of lipoproteins is thought to be an important early step in atherogenesis. The measurement of lipid peroxidation in low-density lipoprotein (LDL) challenged with Cu2+ has become a widespread test to determine the “susceptibility” of LDL to oxidation. The determination of lag time to oxidation is thought to be a measure of the total antioxidant capacity of the LDL. However, we and others have failed to observe any correlation between lag time and the LDL content of its major lipid antioxidant, α-tocopherol. In fact, several studies now suggest a pro-oxidant role for tocopherol under some conditions of LDL oxidation. In the present study we sought to determine if there was a relationship between Cu2+ reduction by LDL and kinetic parameters of LDL oxidation. LDL (0.3 mmol/l cholesterol, ∼0.1 mg protein/ml) was incubated at 30°C with 2 μM Cu2+ and the formation of conjugated dienes measured over a 4-h period. Using neocuproine, an indicator molecule that specifically complexes Cu+ but not Cu2+, the reduction of Cu2+ by LDL was monitored. The final Cu concentration in these assays was 100 μM and neocuproine 750 μM. Cu+ formation was measured by absorbance at 454 nm. A strong negative correlation was observed between copper reduction by LDL and lag time to oxidation (r = −0.66, p < .005, n = 16). Further experiments showed that (1) LDL was able to reduce Cu2+ to Cu+ in a time and concentration-dependent manner; (2) blocking of free —SH groups on LDL apoprotein B by preincubation with dithionitrobenzoic acid (DTNB) had no significant effect on the rate and extent of Cu2+ reduction; (3) consumption of tocopherol in LDL undergoing oxidation with Cu was very rapid (rate = 6 × 10−10 M s−1). When Cu+ formed during incubation with LDL was complexed with neocuproine, there was significant inhibition of LDL oxidation, as indicated by lipid peroxide formation and mobility on agarose gel electrophoresis. Surprisingly, tocopherol consumption was even more rapid in the presence of neocuproine, consistent with a shift in Cu2+/ Cu+ equilibrium and faster reduction of Cu2+ by α-tocopherol. These results indicate that under these conditions tocopherol is a major reducing agent in LDL, converting Cu2+ to Cu+, and therefore, may play an important role in promoting LDL oxidation. However, there was no correlation between LDL tocopherol content and reduction of Cu2+. Examination of the time course of Cu2+ reduction in tocopherol enriched and depleted LDL indicates that tocopherol may determine Cu reduction at early time points but that the eventual capacity of LDL to reduce Cu may depend on more complex interactions between tocopherol and other LDL components.

The Interaction of Resveratrol with Ferrylmyoglobin and Peroxynitrite; Protection Against LDL Oxidation

Resveratrol (3,4',5-trihydroxystilbene) is a natural phytoalexin synthesized in response to injury or fungal attack, found in the grape skin and wine, specially red wine. A large number of studies have demonstrated that resveratrol regulates many biological activities, namely protection against atherosclerosis by a set of pharmacological properties, including the antioxidant activity. In this study, we explored the capacity of resveratrol in protecting low density lipoproteins (LDL) against either ferrylmyoglobin- or peroxynitrite-mediated oxidation and the underlying mechanisms of its antioxidant potential. Resveratrol efficiently decreases the accumulation of hydroperoxides in LDL promoted by ferrylmyoglobin, a potent oxidant formed by the reaction of metmyoglobin with hydrogen peroxide, in a concentration-dependent manner, promptly reducing the oxoferryl complex to metmyoglobin. Simultaneously, resveratrol is consumed as detected by the rapid decrease in the characteristic peak at 310 nm, in a similar way to that observed upon its reaction with peroxidase/H 2 O 2 , pointing to a mechanism of one-electron oxidation and subsequent resveratrol dimer formation. On the other hand, resveratrol inhibits LDL apoprotein modifications induced by peroxynitrite, another potent oxidant formed by the reaction between superoxide and nitric oxide, as assessed by the decrease in apo-B net charge alterations and in carbonyl groups formation mediated by that oxidant. Resveratrol also interacts with peroxynitrite in a similar way to that observed with laccases, suggesting a mechanism of resveratrol oxidation rather than a nitration one. These mechanisms are discussed. Considering that either ferrylmyoglobin or peroxynitrite are physiologically relevant oxidants implicated in several pathologies, including atherosclerosis, our results certainly contribute to the understanding of the antioxidant action of resveratrol and consequently provide a new approach for the cardiovascular benefits associated with moderate consumption of red wine.

The Apoprotein is the Preferential Target for Peroxynitrite-induced LDL Damage Protection by Dietary Phenolic Acids

Peroxynitrite has been shown to modify low-density lipoproteins (LDL) into a form recognized by the macrophage scavenger receptor, suggesting that it may play a significant role in atherogenesis. Considering that the mechanisms underlying LDL modifications by this agent have not been well elucidated, the aim of this study was to characterize the chemical modifications of either the lipid or the protein moieties mediated by synthesized peroxynitrite (preformed) or formed in situ by SIN-1, and evaluate the protective effects of some dietary phenolic acids. Preformed peroxynitrite does not induce LDL lipid peroxidation, as assessed either by formation of conjugated diene isomers or degradation of fatty acids and cholesteryl esters, although a rapid loss of f -tocopherol content occurs. Also, peroxynitrite formed in situ induces only a slight lipid oxidation. In contrast, under conditions where the LDL lipid moiety is not significantly oxidized, peroxynitrite either preformed or formed in situ rapidly elicit significant LDL apoprotein modifications, as evaluated by an increase in carbonyl groups formation and by great decrease in intrinsic tryptophan and thiol groups, in a concentration-dependent manner, that are accompanied by an increase in the LDL net negative charge, leading to an increase in electrophoretic mobility. Phenolic acids, namely caffeic, chlorogenic and ferulic, inhibit all these processes in a concentration dependent way, being the catechols the most efficient. UV spectral analysis of phenols upon interaction with peroxynitrite suggest that, in our assay conditions, such protection is related with the scavenging of this agent by either electron donation for the catechols, caffeic and chlorogenic acids, or nitration for the monophenol ferulic acid. Our data point that in contrast with other physiological oxidants, as ferrylmyoglobin or copper, peroxynitrite triggers the rapid damage to LDL primarily by protein and not lipid oxidation, and that such process is inhibited by dietary phenolic derivatives of cinnamic acids.

Inter-relationships between small, dense low-density lipoprotein (LDL), plasma triacylglycerol and LDL apoprotein B in an atherogenic lipoprotein phenotype in free-living subjects

Inter-relationships between small, dense low-density lipoprotein (LDL), plasma triacylglycerol and LDL apoprotein B in an atherogenic lipoprotein phenotype in free-living subjects

Inter-relationships between small, dense low-density lipoprotein (LDL), plasma triacylglycerol and LDL apoprotein B in an atherogenic lipoprotein phenotype in free-living subjects

A predominance of small, dense low-density lipoprotein (LDL) is a major component of an atherogenic lipoprotein phenotype, and a common, but modifiable, source of increased risk for coronary heart disease in the free-living population. While much of the atherogenicity of small, dense LDL is known to arise from its structural properties, the extent to which an increase in the number of small, dense LDL particles (hyper-apoprotein B) contributes to this risk of coronary heart disease is currently unknown. This study reports a method for the recruitment of free-living individuals with an atherogenic lipoprotein phenotype for a fish-oil intervention trial, and critically evaluates the relationship between LDL particle number and the predominance of small, dense LDL. In this group, volunteers were selected through local general practices on the basis of a moderately raised plasma triacylglycerol (triglyceride) level (>1.5 mmol/l) and a low concentration of high-density-lipoprotein cholesterol (<1.1 mmol/l). The screening of LDL subclasses revealed a predominance of small, dense LDL (LDL subclass pattern B) in 62% of the cohort. As expected, subjects with LDL subclass pattern B were characterized by higher plasma triacylglycerol and lower high-density lipoprotein cholesterol (<1.1 mmol/l) levels and, less predictably, by lower LDL cholesterol and apoprotein B levels (P<0.05; LDL subclass A compared with subclass B). While hyper-apoprotein B was detected in only five subjects, the relative percentage of small, dense LDL-III in subjects with subclass B showed an inverse relationship with LDL apoprotein B (r=-0.57; P<0.001), identifying a subset of individuals with plasma triacylglycerol above 2.5 mmol/l and a low concentration of LDL almost exclusively in a small and dense form. These findings indicate that a predominance of small, dense LDL and hyper-apoprotein B do not always co-exist in free-living groups. Moreover, if coronary risk increases with increasing LDL particle number, these results imply that the risk arising from a predominance of small, dense LDL may actually be reduced in certain cases when plasma triacylglycerol exceeds 2.5 mmol/l.

Detection of Oxidized Low-Density Lipoproteins Using Surface Plasmon Resonance

Management of atherosclerosis is a high priority target. If this is to be achieved, the early detection of risk and risk factors are paramount and integrated with this is a need for the detection of the oxidation state of a patient's low density lipoprotein (LDL). Presently no readily usable technique exists for their rapid determination and in order to develop such a technique a monitoring system must be devised which distinguishes a parameter which changes on oxidation and distinguishes critical and noncritical oxidation products. The strategy which is investigated here is based on the use of a heparin-modified Au-surface plasmon resonance (SPR) device as a modulator of LDL binding, according to its oxidation state. Heparin is strongly negatively charged and seven binding sites for heparin have been identified on the LDL apoprotein consisting of arginine and lysine clusters; these are regarded as identical to the LDL receptor binding sites. The heparin-modified surface was calibrated for LDL and a calibration factor of 1.84 × 10(9) particles mm(-)(2) Δ(o)(-)(1) SPR and instrumental resolution of 9 × 10(6) particles mm(-)(2) obtained which gives sufficient scope to distinguish LDL dependent binding. LDL oxidation could involve the protein and/or lipoprotein, the latter being of interest for athersclerosis risk and the LDL binding to heparin was shown to decrease with degree of protein oxidation as determined by the free amino groups (fluorescamine assay), but was not influenced by lipid oxidation (determined by thiobarbituric acid reactive substances assay, TBARS). The SPR based assay was tested for LDL in plasma and the calibration found to follow that obtained in buffer, although the scatter was higher, probably due to interference from other plasma species. Nevertheless, in the context of the normal distribution of LDL in healthy patients, the assay would almost certainly be able to determine Ox-LDL in atherosclerotic patients.

The interaction of lower alcohols with apoB in spin labeled human plasma low density lipoproteins (LDL)

In this study the interaction of alcohol with the macromolecular lipid–protein assembly represented by human plasma low density lipoproteins (LDL) was investigated. The spin label which covalently binds to the side chain amino group of lysines as well as terminal amino groups was attached to the spin labeled apoprotein (apoB) of native LDL in order to observe the protein component in the electron spin resonance (ESR) spectrum. The interaction of different lower alcohols (methanol, ethanol, propanol and butanol) with the spin labeled LDL was studied for two alcohol concentrations (0.3 and 3.0 M). The ESR spectra indicate a decrease of the hyperfine splitting and narrowing of the linewidth upon the action of alcohol that leads to the conclusion that alcohol provokes a change in the apoB conformation. These findings are explained by following the arguments of the phospholipid mediated mechanism of alcohol action, through the modulation of the lipid packing free volume which results in the protein conformational change.

Guggulsterone, a potent hypolipidaemic, prevents oxidation of low density lipoprotein

The oxidation of low density lipoprotein (LDL) induced by Cu+2 caused marked oxidative changes in the lipid and protein constituents of this lipoprotein in vitro. Guggulsterone prevented the generation of lipid peroxides measured as thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes. This compound protected LDL against depletion of lipid constituents such as cholesterol, cholesterol esters, triglyceride and phospholipids as well as inhibiting the conversion of cholesterol into oxygenated cholesterols. Oxidized LDL containing less apoprotein B with a high protein carbonyl value, was more electronegative, as evidenced by the increase in relative electrophoretic mobility (REM) on agarose gel. Guggulsterone significantly protected LDL apoprotein as measured by reversal of REM after oxidation. The protective action of guggulsterone may be due to its free radical scavenging property as this compound significantly inhibited the generation of hydroxyl radicals in a nonenzymic system. © 1997 John Wiley & Sons, Ltd.