Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes

The alteration of structural and biological properties of human plasma low density lipoprotein (LDL) exposed to oxidative conditions is in part ascribed to lipid peroxidation. The objective of this investigation was to measure quantitatively several parameters in oxidizing LDL indicative for lipid peroxidation. Exposure of freshly prepared EDTA-free LDL to an oxygen-saturated buffer led to a complete depletion of alpha- and gamma-tocopherol within 6 hr, thereafter lipid peroxidation commenced as indicated by the kinetics of the loss of linoleic (18:2) and arachidonic (20:4) acids, the formation of aldehydic lipid peroxidation products and fluorescent apoB. Within 24 hr of oxidation, on average 79 nmol of 18:2 (initial 345) and 12.8 nmol of 20.4 (initial 25.6) were oxidized per mg of LDL and the sample contained in total 7.1 nmol of aldehydes with the following molar distribution: 36.6% malonaldehyde, 25% hexanal, 8.9% propanal, 8.2% 4-hydroxynonenal, 7.6% butanal, 4.1% 2.4-heptadienal, 3.4% pentanal, 3.4% 4-hydroxyhexenal, and 2.5% 4-hydroxyoctenal. Malonaldehyde was predominantly (93%) in the aqueous phase, whereas the other aldehydes remained mostly (34-98%) within the LDL particle, where the total aldehyde concentration was in the range of 12 mM. Oxidized LDL exhibited a 1.6-fold enhanced electrophoretic mobility. Similarily, native LDL incubated for 5 hr with aldehydes showed increased electrophoretic mobility. At equal concentrations (5 mM) 4-hydroxynonenal was most effective, followed by 2,4-heptadienal, hexanal, and malonaldehyde. This study reports for the first time the rate and extent of the change of LDL constituents occurring during lipid peroxidation.     

The effect of histidine modification on copper-dependent lipid peroxidation in human low-density lipoprotein

Summary

Lipid peroxidation and subsequent oxidative modification of low-density lipoprotein (LDL) have been implicated as causal events in atherosclerosis. Cu²⁺ may play an important role in LDL oxidation by binding to histidine residues of apolipoprotein B-100 (apo B) and initiating and propagating lipid peroxidation. To investigate the role of histidine residues, we used diethylpyrocarbonate (DEPC), a lipid-soluble histidine-specific modifying reagent. When LDL (0.1 mg protein/ml, or 0.2 µM) was incubated with DEPC (1 mM), at least 76 ± 7% of the histidine residues in apo B were modified. Treatment of LDL with DEPC led to an increase in the rate of Cu²⁺-induced initiation of lipid peroxidation (R_i), but a significant decrease in the rate of propagation. These changes resulted in an overall increased resistance of LDL to oxidation, with a significantly increased lag phase preceding the propagation phase of lipid peroxidation. In contrast to DEPC, ascorbate completely prevented the initiation of LDL oxidation (R_i = 0). Our data indicate that there are two types of copper/histidine binding sites on apo B: those facing the lipid core of the LDL particle, which mediate the propagation of lipid peroxidation and are modified by DEPC; and those found on the surface of the LDL particle exposed to the aqueous environment, which are responsible for mediating the initiation of lipid peroxidation and are modifiable by ascorbate in the presence of Cu²⁺.     

Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions.

The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+----CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.     

Aldehyde adducts generated during lipid peroxidation modification of proteins

Abstract

Various lines of evidence indicate that an important part in the pathogenesis of atherosclerosis is the modification of the plasma low-density lipoproteins (LDLs). A large number of pro-inflammatory and pro-atherogenic properties have been ascribed to the oxidatively modified LDLs and their components. There is considerable evidence to support the role of lipid peroxidation products, reactive aldehydes in particular, originating from the oxidized LDL as important signaling molecules in the context of the atherosclerotic lesion. These aldehydes generated during the peroxidation of LDL exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts on the apolipoprotein B-100 particle in LDL. Characterization of the aldehyde adducts generated in the protein is therefore critical in understanding the nature of the oxidized LDL. However, the majority of adducts generated during the oxidative modification of LDL have not yet been chemically characterized. In this review, the current status of aldehyde adducts quantitatively analyzed in the Cu²⁺-oxidized LDL is reviewed.     

Detection of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice

Lipid peroxidation in cellular membranes leads to the formation of toxic aldehydes. One product provided with particular reactivity has been identified as 4-hydroxynonenal and thoroughly studied as one of the possible mediators of the cellular injury induced by pro-oxidants. In the present study we have searched for the presence of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice, since under this experimental condition the level of lipid peroxidation is much greater than in the case of CCl4 or BrCCl3 hepatotoxicity. 4-Hydroxynonenal was looked for in liver extracts as either free aldehyde or its 2,4-dinitrophenylhydrazone derivative. In both cases, by means of thin-layer chromatography (TLC) and high-pressure liquid chromatography, a well resolved peak corresponding to the respective standards (free aldehyde or 2,4-dinitrophenylhydrazone derivative) was obtained. Total carbonyls present in the liver of intoxicated animals were detected as 2,4-dinitrophenylhydrazone derivatives. The hydrazones were pre-separated by TLC into three fractions according to different polarity (polar, non-polar, fraction I, and non-polar, fraction II). The amounts of carbonyls present in each fraction were determined by ultraviolet-visible spectroscopy. 'Non-polar carbonyls, fraction II' were further fractionated by TLC. The fraction containing alkanals and alk-2-enals was analyzed by high-pressure liquid chromatography and several aldehydes were identified. In addition, protein bound carbonyls were determined in the liver of bromobenzene-treated mice. The biological implications of the finding of 4-hydroxynonenal and other carbonyls in vivo in an experimental model of hepatotoxicity are discussed.     

Tetravalent vanadium mediated oxidation of low density lipoprotein

1. Tetravalent vanadium causes oxidation of low density lipoprotein (LDL) as manifest by protein degradation and lipid peroxidation. 2. Oxidative modification of the apolipoprotein B-100 is paralleled by the formation of thiobarbituric acid reactive substance and fluorescent chromolipid production. 3. The metal chelators ethylenediamine tetracetic acid and desferrioxamine, and the alcohols, ethanol and isopropanol inhibit the oxidation of LDL by tetravalent vanadium. No inhibition is observed with superoxide dismutase, catalase or mannitol. 4. The data suggest that aldehydes formed during the process of lipid peroxidation induced by tetravalent vanadium react with the proteins in LDL to form fluorescent chromolipids and that the oxidative process originates within the hydrophobic domain of LDL.     

Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cell. I. Chemical modifications of ultraviolet-treated low-density lipoproteins

A new experimental model system constituted by ultraviolet-treated low-density lipoproteins (LDL) has been designed in order to investigate the biological effects of lipid peroxides entering the cell through the endocytotic pathway. This paper reports the chemical modifications of the lipid components and apolipoproteins of the ultraviolet-treated LDL. Human LDL were submitted to short ultraviolet radiations (254 nm, 0.5 mW/cm2, for variable periods of time) and compared to LDL peroxidized by iron. The lipid peroxidation was monitored by following the formation of the peroxidation products (conjugated dienes, thiobarbituric acid-reactive substances (TBARS) and fluorescent lipid-soluble products) and the change of the composition in polyunsaturated fatty acids, carotenes and vitamin E. Several parameters of the apo B-100 structure were investigated: molecular size (by SDS-PAGE) and TNBS-reactive amino groups (chemical determination by trinitrobenzene sulfonic acid). The most important feature was the absence of major modification of apo B-100 in ultraviolet-treated LDL: the molecular weight and the content in TNBS-reactive amino groups of apo B-100 were not modified. In contrast, iron-treated LDL exhibited a loss of the apo B-100 band and a decrease in the number of TNBS-reactive amino group. Both ultraviolet radiations and iron ions induced a significant decrease in the content of polyunsaturated fatty acids, carotenes and vitamin E together with a large formation of lipid peroxidation products. However, the time-course of the formation of conjugated dienes, TBARS and fluorescent lipid-soluble products was quite different using the two oxidative systems. These results demonstrate that ultraviolet radiations induced a strong peroxidation of the lipid content of LDL and no (or only minor) changes in the apolipoprotein moiety whereas iron-catalyzed peroxidation resulted in the formation fo lipid peroxidation products as well as apo B alterations.     

Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS

Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.     

Iron-ascorbate alters the efficiency of Caco-2 cells to assemble and secrete lipoproteins

Although oxidative stress has been implicated in development of gut pathologies, its role in intestinal fat transport has not been investigated. We assessed the effect of Fe(2+)-ascorbate-mediated lipid peroxidation on lipid synthesis, apolipoprotein biogenesis, and lipoprotein assembly and secretion. Incubation of postconfluent Caco-2 cells with iron(II)-ascorbate (0.2 mM/2 mM) in the apical compartment significantly promoted malondialdehyde formation without affecting sucrase activity, transepithelial resistance, DNA and protein content, and cell viability. However, addition of the oxygen radical-generating system reduced 1) [(14)C]oleic acid incorporation into cellular triglycerides (15%, P < 0.0002) and phospholipids (16%, P < 0.0005); 2) de novo synthesis of cellular apolipoprotein A-I (apo A-I) (18%, P < 0.05), apo A-IV (38%, P < 0.05), and apo B-48 (45%, P < 0.003) after [(35)S]methionine addition; and 3) production of chylomicrons (50%), VLDL (40%), LDL (37%), and HDL (30%) (all P < 0.0001). In contrast, increased total cellular cholesterol formation (96%, P < 0.0001), assayed by [(14)C]acetate incorporation, was noted, attributable to marked elevation (70%, P < 0.04) in activity of DL-3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. The ratio of Acyl-CoA to cholesterol acyltransferase, the esterifying cholesterol enzyme, remained unchanged. Fe(2+)-ascorbate-mediated lipid peroxidation modifies intracellular fat absorption and may decrease enterocyte efficiency in assembling and transporting lipids during gut inflammation.     

Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells

Hepatic oxidative stress and lipid peroxidation are common features of several prevalent disease states, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), a common component of the metabolic syndrome. These conditions are characterized in part by excessive accumulation of lipids within hepatocytes, which can lead to autocatalytic degradation of cellular lipids giving rise to electrophilic end products of lipid peroxidation. The pathobiology of reactive lipid aldehydes remains poorly understood. We therefore sought to investigate the effects of 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) on the transport and secretion of very low-density lipoprotein using HepG2 cells as a model hepatocyte system. Physiologically relevant concentrations of 4-HNE and 4-ONE rapidly disrupted cellular microtubules in a concentration-dependent manner. Interestingly, 4-ONE reduced apolipoprotein B-100 (ApoB) secretion while 4-HNE did not significantly impair secretion. Both 4-HNE and 4-ONE formed adducts with ApoB protein, but 4-HNE adducts were detectable as mono-adducts, while 4-ONE adducts were present as protein–protein cross-links. These results demonstrate that reactive aldehydes generated by lipid peroxidation can differ in their biological effects, and that these differences can be mechanistically explained by the structures of the protein adducts formed.     

Investigation of Lipid Peroxidation in Human Low Density Lipoprotein

Human plasma low density lipoprotein (LDL) exposed to oxygen saturated buffer becomes depleted of alpha-tocopherol within 3 to 6 hours. Thereafter, lipid peroxidation commences as evidenced by the loss of 18:2 (67nmol/mg LDL) and 20:4 (12nmol/mg LDL) and the concomitant formation of 4-hydroxy-nonenal (0.28 nmol/mg LDL) and fluorescent compounds. The major fluorophor in apo B of oxidized LDL has an excitation maximum at 355 nm and an emission maximum at 430 nm. A fluorophor with the same spectral properties is produced in apo B, if LDL is incubated with 4-hydroxynonenal, whereas malonal-dehyde gives a fluorophor with excitation and emission maxima at 400/470nm. Three-dimensional fluorescence spcetroscopy proved to be an useful tool in analysing the complex fluorescence of apo B.     

Expression and synthesis of TGFbeta1 is induced in macrophages by 9-oxononanoyl cholesterol, a major cholesteryl ester oxidation product

Within the broad variety of compounds generated via oxidative reactions in low density lipoproteins (LDL) and subsequently found in the atherosclerotic plaque, are aldehydes still esterified to the parent lipid and termed core-aldehydes. The most represented cholesterol core-aldehyde in LDL is 9-oxononanoyl cholesterol (9-ONC), an oxidation product of cholesteryl linoleate. Here we report that 9-ONC, at concentration actually detectable in biological material, significantly up-regulates the expression and the synthesis of the pro-fibrogenic cytokine transforming growth factor beta1 (TGFbeta1) by cultured macrophages. As previously demonstrated for other lipid oxidation products present in LDL, namely a biologically representative mixture of oxysterols and the unesterified aldehyde 4-hydroxynonenal, these effects on TGFbeta1 by 9-ONC further points to LDL lipid oxidation as a powerful source of pro-fibrogenic stimuli.     

Monoclonal Antibodies for Detection of 4-Hydroxynonenal Modified Proteins

A promising approach to study lipid peroxidation pathology is antibodies recognizing aldehydes which react with and became bound to amino acid side chains of proteins. We present in this study the characterization of several monoclonal antibodies which recognize 4-hydroxynonenal (HNE) modified proteins. Six out of 20 antibodies recognizing HNE modified BSA were able to detect HNE-protein adducts in peroxidized liver microsomes. Two of these antibodies were selected and characterized. Both antibodies could also detect HNE-protein adducts in oxidized low density lipoprotein. They exhibit no detectable cross reaction with proteins modified by malonaldehyde, nonanal, nonenal and 4-hydroxyhexenal. Protein bound 4-hydroxyoctenal and 4-hydroxydecenal were recognized to some extent. Further characterization revealed that the two antibodies are highly selective for HNE bound to histidine with only some cross reaction to HNE bound to lysine and cysteine. Preliminary quantitative ELISA-analysis showed that oxidized microsomes and oxidized LDL contain 12 nmol and 3 nmol HNE-histidine per mg protein respectively.     

Direct evidence for apo B-100-mediated copper reduction: studies with purified apo B-100 and detection of tryptophanyl radicals

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.     

Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation

The mechanism of the formation of 4-hydroxynonenal through the NADPH-linked microsomal lipid peroxidation was investigated. The results were as follows: 4-hydroxynonenal arises exclusively from arachidonic acid contained in the polar phospholipids, neither arachidonic acid of the neutral lipids nor linoleic acid of the polar or neutral lipids are substrates for 4-hydroxynonenal generation. This finding results from the estimation of the specific radioactivity of 4-hydroxynonenal produced by microsomes prelabelled in vivo with [U-14C]arachidonic acid. Phospholipid-bound 15-hydroperoxyarachidonic acid would have the structural requirements needed for 4-hydroxynonenal (CH3-(CH2)4-CH(OH)-CH=CH-CHO). Microsomes supplemented with 15-hydroperoxyarachidonic acid and NADPH, ADP/iron converted only minimal amounts (0.6 mol%) of 15-hydroperoxyarachidonic acid into 4-hydroxynonenal; similarly, 15-hydroperoxyarachidonic acid incubated at pH 7.4 in the presence of ascorbate/iron yielded only small amounts of 4-hydroxynonenal with a rate orders of magnitude below that observed with microsomes. Phospholipid-bound 15-hydroperoxyarachidonic acid is therefore not a likely intermediate in the reaction pathway leading to 4-hydroxynonenal. The rate of 4-hydroxynonenal formation is highest during the very initial phase of its formation and the onset does not show a lag phase, suggesting a transient intermediate predominantly formed during the early phase of microsomal lipid peroxidation. After 60 min of incubation, 204 nmol polyunsaturated fatty acids (20 nmol 18:2, 143 nmol 20:4, 41 nmol 22:6) were lost per mg microsomal protein and the incubation mixture contained 206 nmol lipid peroxides, 71.6 nmol malonic dialdehyde and 4.6 nmol 4-hydroxynonenal per mg protein. Under artificial conditions (pH 1.0, ascorbate/iron, 20 h of incubation) not comparable to the microsomal peroxidation system, 15-hydroperoxyarachidonic acid can be decomposed in good yields (15 mol%) into 4-hydroxynonenal. Autoxidation of arachidonic acid in the presence of ascorbate/iron gave after 25 h of incubation 2.8 mol% (pH 7.4) and 1.5 mol% (pH 1.0) 4-hydroxynonenal. The most remarkable difference between the non-enzymic system and the enzymic microsomal system is that the latter forms 4-hydroxynonenal at a much higher rate.     

Mechanisms of oxidative modification of low density lipoproteins under conditions of oxidative and carbonyl stress

Low-molecular-weight aldehydes (glyoxal, methylglyoxal, 3-deoxyglucosone) generated on autooxidation of glucose under conditions of carbonyl stress react much more actively with amino groups of L-lysine and epsilon-amino groups of lysine residues of apoprotein B-100 in human blood plasma low density lipoproteins (LDL) than their structural analogs (malonic dialdehyde (MDA), 4-hydroxynonenal) resulting on free radical oxidation of lipids under conditions of oxidative stress. Glyoxal-modified LDL aggregate in the incubation medium with a significantly higher rate than LDL modified by MDA, and MDA-modified LDL are markedly more poorly absorbed by cultured human macrophages and significantly more slowly eliminated from the rat bloodstream upon intravenous injection. Studies on kinetics of free radical oxidation of rat liver membrane phospholipids have shown that ubiquinol Q(10) is the most active lipid-soluble natural antioxidant, and suppression of ubiquinol Q(10) biosynthesis by beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors (statins) is accompanied by intensification of lipid peroxidation in rat liver biomembranes and in LDL of human blood plasma. Injection of ubiquinone Q(10) protects the human blood plasma LDL against oxidation and prevents oxidative stress-induced damages to rat myocardium. A unified molecular mechanism of atherogenic action of carbonyl-modified LDL in disorders of lipid and carbohydrate metabolism is discussed.     

Presence of two forms of apolipoprotein B in patients with dyslipoproteinemia

Current information suggests that the major forms of the human B apolipoproteins, B-100 and B-48, are under separate genetic control and are synthesized by the liver and intestine, respectively. The apolipoprotein B composition of plasma lipoproteins has been determined in order to gain insight into the metabolic defects in patients with dyslipoproteinemias. Patients with type I and type V hyperlipoproteinemia can be separated into two groups based on apolipoprotein composition and triglyceride concentration. The first group had markedly elevated plasma triglycerides with B-48 in the 1.006 g/ml density fraction and only B-100 within IDL and LDL. The second group had plasma triglycerides less than 1200 mg % and only B-100 in all density fractions. Patients with type III hyperlipoproteinemia had B-48 in only the density less than 1.006 g/ml with B-100 in IDL and LDL; the type III hyperlipoproteinemic patient with apolipoprotein E deficiency, however, had B-48 in density less than 1.006 g/ml fraction, IDL, and LDL. Patients with type IIa, IIb, and IV hyperlipoproteinemia had only B-100 in all density fractions. These combined results are interpreted as indicating that B-48 is associated with triglyceride-rich lipoproteins synthesized by the intestine and that patients with phenotypes I, III, and V have defects in chylomicron remnant metabolism. In addition, in patients with types I and V hyperlipoproteinemia, mild hypertriglyceridemia appears to be associated with lipoprotein particles of liver origin.     

Effects of oolonghomobisflavan A on oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been suggested to be involved in the onset of atherosclerosis. Oolong tea contains unique polyphenols including oolonghomobisflavan A (OFA). In this study, the effects of OFA on LDL oxidation by ROS and RNS were investigated in vitro. OFA suppressed formation of cholesterol ester hydroperoxides in LDL oxidized by peroxyl radical and peroxynitrite, and formation of thiobarbituric acid reactive substances in LDL oxidized by Cu²⁺. In addition, OFA inhibited fragmentation, carbonylation, and nitration of apolipoprotein B-100 (apo B-100) in the oxidized LDL, in which heparin-binding activity of apo B-100 was protected by OFA. Our results suggest that OFA exhibits antioxidant activity against both lipid peroxidation and oxidative modification of apo B-100 in LDL oxidized by ROS and RNS. Polyphenols in oolong tea may prevent atherosclerosis by reducing oxidative stress.     

Comparison of the Inactivation of Microsomal Glucose-6-Phosphatase by in Situ Lipid Peroxidation-Derived 4-Hydroxynonenal and Exogenous 4-Hydroxynonenal

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied.

2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes.

3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E.

4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5μM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150μM or 300μM was needed with a preincubation time of 30 and 60 min, respectively.

5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynbnenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.     

Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low-density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoprotein.

4-Hydroxynonenal (HNE) is a major aldehydic propagation product formed during peroxidation of unsaturated fatty acids. The aldehyde was used to modify freshly prepared human low-density lipoprotein (LDL). A polyclonal antiserum was raised in the rabbit and absorbed with freshly prepared LDL. The antiserum did not react with human LDL, but reacted with CuCl2-oxidized LDL and in a dose-dependent manner with LDL, modified with 1, 2 and 3 mM-HNE, in the double-diffusion analysis. LDL treated with 4 mM of hexanal or hepta-2,4-dienal or 4-hydroxyhexenal or malonaldehyde (4 or 20 mM) did not react with the antiserum. However, LDL modified with 4 mM-4-hydroxyoctenal showed a very weak reaction. Lipoprotein (a) and very-low-density lipoprotein were revealed for the first time to undergo oxidative modification initiated by CuCl2. This was evidenced by the generation of lipid hydroperoxides and thiobarbituric acid-reactive substances, as well as by a marked increase in the electrophoretic mobility. After oxidation these two lipoproteins also reacted positively with the antiserum against HNE-modified LDL.