Georg Waeg

Role of vitamin E in preventing the oxidation of low-density lipoprotein.

The fatty acid composition, antioxidants, and the oxidation resistance of the low-density lipoproteins (LDL) from a number of different donors were determined. The oxidation resistance of LDL, as determined in vitro by the duration of the lag-phase in copper ion-induced oxidation, did not correlate with the alpha-tocopherol content of the LDL. By supplementating plasma with vitamin E, the alpha-tocopherol content of LDL could be increased from approximately 9 to 30 mol/mol LDL and also the oxidative resistance increased nearly linearly with increasing alpha-tocopherol content. The results indicate that alpha-tocopherol is an important, yet not the only parameter that determines the oxidation resistance of LDL.

Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfunction in synaptosomes by amyloid β-peptide : Role of the lipid peroxidation product 4-hydroxynonenal

Abstract: Deposits of amyloid β‐peptide (Aβ), reduced glucose uptake into brain cells, oxidative damage to cellular proteins and lipids, and excitotoxic mechanisms have all been suggested to play roles in the neurodegenerative process in Alzheimers disease. Synapse loss is closely correlated with cognitive impairments in Alzheimers disease, suggesting that the synapse may be the site at which degenerative mechanisms are initiated and propagated. We report that Aβ causes oxyradical‐mediated impairment of glucose transport, glutamate transport, and mitochondrial function in rat neocortical synaptosomes. Aβ induced membrane lipid peroxidation in synaptosomes that occurred within 1 h of exposure; significant decreases in glucose transport occurred within 1 h of exposure to Aβ and decreased further with time. The lipid peroxidation product 4‐hydroxynonenal conjugated to synaptosomal proteins and impaired glucose transport; several antioxidants prevented Aβ‐induced impairment of glucose transport, indicating that lipid peroxidation was causally linked to this adverse action of Aβ. FeSO4 (an initiator of lipid peroxidation), Aβ, and 4‐hydroxynonenal each induced accumulation of mitochondrial reactive oxygen species, caused concentration‐dependent decreases in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction, and reduced cellular ATP levels significantly. Aβ also impaired glutamate transport, an effect blocked by antioxidants. These data suggest that Aβ induces membrane lipid peroxidation, which results in impairment of the function of membrane glucose and glutamate transporters, altered mitochondrial function, and a deficit in ATP levels; 4‐hydroxynonenal appears to be a mediator of these actions of Aβ. These data suggest that oxidative stress occurring at synapses may contribute to the reduced glucose uptake and synaptic degeneration that occurs in Alzheimers disease patients. They further suggest a sequence of events whereby oxidative stress promotes excitotoxic synaptic degeneration and neuronal cell death in a variety of different neurodegenerative disorders.

4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes

Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimers, Parkinsons and Huntingtons diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4-hydroxynonenal potentiates oxidative cascades. Collectively, these findings suggest that 4-hydroxynonenal plays important roles in oxidative impairment of synaptic functions that would be expected to promote excitotoxic cascades.

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimers disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β‐peptide (Aβ). Aβ‐associated free radicals cause lipid peroxidation, a major product of which is 4‐hydroxy‐2‐trans‐nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein‐specific spin label, MAL‐6(2,2,6,6‐tetramethyl‐4‐maleimidopiperidin‐1‐oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid‐specific stearic acid label, 5‐nitroxide stearate, was used to probe for HNE‐induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration‐ and time‐dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque‐ and Aβ‐rich AD hippocampal and inferior parietal brain regions. HNE‐induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE‐caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µM). The results obtained are discussed with relevance to the hypothesis of Aβ‐induced free radical‐mediated lipid peroxidation, leading to subsequent HNE‐induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.

Effect of Antioxidants on Oxidative Modification of LDL

Human low density lipoprotein (LDL) with a molecular mass of 2.5 million contains on average 1300 molecules of polyunsaturated fatty acids (PUFAs) bound in the different lipid classes. The predominant antioxidant in LDL is alpha-tocopherol, with an average of 6 molecules in each LDL particle. The other substances with potential antioxidant activity are: gamma-tocopherol, beta-carotene, alpha-carotene, lycopene, cryptoxanthin, cantaxanthin, phytofluene and ubiquinol-10. Each is present in amounts of only 1/20th to 1/300th of that of alpha-tocopherol. If LDL is exposed to oxidative conditions (Cu++ ions, macrophages) a lag phase precedes the oxidation of PUFAs. During the lag phase the antioxidants disappear with alpha-tocopherol the first to go and beta-carotene the last. The lag phase, which can readily be determined, is an index of the oxidation resistance of LDL. If LDL is loaded with vitamin E in vitro its oxidation resistance increases linearly with its alpha-tocopherol content according to the equation, y = kx+a. The same relationship is applicable if the alpha-tocopherol content of LDL is increased by taking oral vitamin E. Daily doses of 150, 225, 800 and 1200 IU RRR-alpha-tocopherol increased the LDL alpha-tocopherol on average to 138%, 158%, 144% and 215% of the initial value, the oxidation resistance being increased to 118%, 156%, 135% and 175%, respectively. The efficiency of vitamin E-dependent (= k) and the vitamin independent (= a) oxidation resistance seem to be subject specific with strong individual variation.(ABSTRACT TRUNCATED AT 250 WORDS)

4‐Hydroxynonenal, a Lipid Peroxidation Product, Rapidly Accumulates Following Traumatic Spinal Cord Injury and Inhibits Glutamate Uptake

Abstract: Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4‐hydroxynonenal (HNE), which cross‐links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein‐bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein‐bound HNE increased in content in the damaged spinal cord at early times following injury (1–24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein‐bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose‐dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms.

4-Hydroxynonenal, a product of lipid peroxidation, inhibits dephosphorylation of the microtubule-associated protein tau.

IN Alzheimers disease (AD) the microtubule-associated protein tau is excessively phosphorylated in degenerating neurons, but the mechanisms underlying the increased phosphorylation are unknown. Recent findings suggest that oxidative stress, and membrane lipid peroxidation in particular, contributes to the neurodegenerative process in AD. We now report that following exposure of cultured rat hippocampal neurons to 4-hydroxy-nonenal (HNE), an aldehydic product of membrane lipid peroxidation, tau is resistant to dephoshorylation. Immunocytochemical and Western blot analyses using phosphorylation-sensitive tau antibodies showed that HNE treatment causes a moderate increase in basal levels of tau phosphorylation, and prevents tau dephosphorylation by alkaline phosphatase in neurons pretreated with the phosphatase inhibitor okadaic acid. Studies with anti-HNE antibodies showed that HNE binds directly to tau, and that HNE immunoreactivity localizes to cell bodies and axons, cell compartments that contain tau. These data suggest a role for HNE in altered tau phosphorylation and neurofibrillary degeneration in AD.

4-Hydroxynonenal, an aldehydic product of lipid peroxidation, impairs signal transduction associated with muscarinic acetylcholine and metabotropic glutamate receptors : Possible action on Gαq/11

Abstract: Considerable data indicate that oxidative stress and membrane lipid peroxidation contribute to neuronal degeneration in an array of age‐related neurodegenerative disorders. In contrast, the impact of subtoxic levels of membrane lipid peroxidation on neuronal function is largely unknown. We now report that 4‐hydroxy‐nonenal (HNE), an aldehydic product of lipid peroxidation, disrupts coupling of muscarinic cholinergic receptors and metabotropic glutamate receptors to phospholipase C‐linked GTP‐binding proteins in cultured rat cerebrocortical neurons. At subtoxic concentrations, HNE markedly inhibited GTPase activity, inositol phosphate release, and elevation of intracellular calcium levels induced by carbachol (muscarinic agonist) and (RS)‐3,5‐dihydroxyphenyl glycine (metabotropic glutamate receptor agonist). Maximal impairment of agonist‐induced responses occurred within 30 min of exposure to HNE. Other aldehydes, including malondialdehyde, had little effect on agonist‐induced responses. Antioxidants that suppress lipid peroxidation did not prevent impairment of agonist‐induced responses by HNE, whereas glutathione, which is known to bind and detoxify HNE, did prevent impairment of agonist‐induced responses. HNE itself did not induce oxidative stress. Immunoprecipitation‐western blot analysis using an antibody to HNE‐protein conjugates showed that HNE can bind to Gαq/11. HNE also significantly suppressed inositol phosphate release induced by aluminum fluoride. Collectively, our data suggest that HNE plays a role in altering receptor‐G protein coupling in neurons under conditions of oxidative stress that may occur both normally, and before cell degeneration and death in pathological settings.

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, nonanal 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.

Oxidation of lipoprotein Lp(a). A comparison with low-density lipoproteins.

Aimed at identifying possible mechanisms of the suggested high atherogenicity of Lp(a), its susceptibility for Cu(II)-induced oxidation was studied and compared with that of LDL. Since the content of antioxidants as well as the fatty acid pattern of a lipoprotein greatly affects its oxidizability, Lp(a) and LDL were characterized first with respect to these substances. Paired samples of low-density lipoproteins (LDL) and Lp(a) were isolated from seven individual donors and compared with each other. This study showed that LDL and Lp(a) are very similar with respect to their fatty acid and antioxidant composition. LDL contains approx. 1132 nmol of total fatty acids/mg lipoprotein and LDL 1466 nmol total fatty acids/mg lipoprotein. Analysis of the fatty acid composition of individual lipid classes (cholesteryl esters, phospholipids and triacylglycerols) revealed also a high similarity in the composition of these lipid classes between the two lipoproteins. A comparison of the antioxidant composition showed that Lp(a) contains less alpha-tocopherol than LDL (1.6 +/- 0.35 nmol/mg vs. 2.1 +/- 0.25 nmol/mg LDL). In copper(II)-induced lipid peroxidation experiments we found a striking difference in the susceptibility of individual lipoprotein classes between all donors. In addition, Lp(a) exhibited a 1.2 to 2.4 longer lag-phase than the corresponding LDL preparation from the same blood donor. Treatment of Lp(a) with neuraminidase resulted in a drastic decrease of the lag-phase of Lp(a). Neuraminidase treatment of LDL on the other hand had no significant effects on its susceptibility to oxidation. Supplementation of neuraminidase-treated Lp(a) with N-acetylneuraminic acid (NANA) at concentrations comparable to the naturally occurring amounts of NANA in the Lp(a) protein moiety led to an increase of the lag-phase yielding values which were comparable to those observed with native Lp(a). These results demonstrate that the fatty acid composition as well as the antioxidant concentrations of Lp(a) and LDL are quite similar; despite this fact, Cu2(+)-mediated oxidation of Lp(a) is retarded in comparison to LDL which might be due to the higher content of NANA in Lp(a).