Advances in methods for the determination of biologically relevant lipid peroxidation products

Abstract

Lipid peroxidation is recognized to be an important contributor to many chronic diseases, especially those of an inflammatory pathology. In addition to their value as markers of oxidative damage, lipid peroxidation products have also been shown to have a wide variety of biological and cell signalling effects. In view of this, accurate and sensitive methods for the measurement of lipid peroxidation products are essential. Although some assays have been described for many years, improvements in protocols are continually being reported and, with recent advances in instrumentation and technology, highly specialized and informative techniques are increasingly used. This article gives an overview of the most currently used methods and then addresses the recent advances in some specific approaches. The focus is on analysis of oxysterols, F₂-isoprostanes and oxidized phospholipids by gas chromatography or liquid chromatography mass spectrometry techniques and immunoassays for the detection of 4-hydroxynonenal.     

Elevated F2-isoprostanes in thalassemic patients

This study was aimed at investigating oxidative stress in thalassemic patients by measurement of the oxidative damage biomarker, F₂-isoprostanes (F₂-IsoPs), using gas chromatography-mass spectrometry. The results showed that the mean value of urinary F₂-IsoPs, normalized with creatinine, in the thalassemic group was significantly higher than that from healthy subjects (3.38 ± 2.15 ng/mg creatinine vs 0.86 ± 0.55 ng/mg creatinine, respectively), and the mean value of plasma total F₂-IsoPs in the thalassemic group was also significantly higher than that from healthy subjects (0.39 ± 0.15 ng/ml vs 0.18 ± 0.03 ng/ml, respectively). Serum ferritin, erythrocyte superoxide dismutase (SOD), glutathione peroxidase, glutathione, and TBARS levels after treatment of erythrocytes with H₂O₂ were also investigated, and serum ferritin and erythrocyte SOD levels were significantly higher in thalassemic patients. Our findings are consistent with oxidative stress in thalassemia patients.     

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein-protein adducts in 113 human colon tumours. The presence of acrolein-protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.     

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein–protein adducts in 113 human colon tumours. The presence of acrolein–protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.     

From a computational point of view: deciphering the molecular synergism between oxidative stress-induced lipid peroxidation products and metabolic dysfunctionality of human liver mitochondrial aldehyde dehydrogenase-2

Accumulation of oxidative stress-induced lipid peroxidation products; 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE), inactivates the metabolic activity of human liver aldehyde dehydrogenase 2 (ALDH2), an enzyme that converts acetaldehyde to carboxylic acids during alcohol metabolism. Previous reports showed that 4HNE and 4ONE covalently target the catalytic Cys302 residue and inactivate ALDH2, thereby preventing the metabolism of acetaldehyde (ACE), its primary substrate. However, the molecular basis of these reactions remains elusive. Therefore, in this study, we investigated the inactivation mechanism of 4HNE and 4ONE on ALDH2 using advanced computational tools. Interestingly, our findings revealed that both inhibitors significantly distorted ALDH2 oligomerization and co-enzyme binding domains, which are crucial to its metabolic activity. The resulting structural alterations could disrupt co-factor binding and enzymatic oligomerization mechanisms. In contrast to the acetaldehyde, 4HNE and 4ONE were bound to ALDH2 with high affinity, coupled with high energy contributions by catalytic site residues and could indicate the possible mechanism by which acetaldehyde is displaced from ALDH2 binding by 4HNE and 4ONE. These findings will be useful in the design of novel compounds that either mop up or block the binding of these endogenous compounds to ALDH2 thereby preventing the development of associated cancers and neurodegenerative diseases.     

Beta-glucan from Saccharomyces cerevisiae reduces plasma lipid peroxidation induced by haloperidol

Since oxidative stress observed in schizophrenia may be caused partially by the treatment of patients with various antipsychotics, the aim of the study was to establish the effects of beta-d-glucan, polysaccharide derived from the yeast cell walls of species such as Saccharomyces cerevisiae, and the antipsychotics (the first generation antipsychotic (FGA) - haloperidol and the second generation antipsychotic (SGA) - amisulpride) action on plasma lipid peroxidation in vitro. Lipid peroxidation in human plasma was measured by the level of thiobarbituric acid reactive species (TBARS). The samples of plasma from healthy subjects were incubated with haloperidol or amisulpride in the presence of beta-glucan (4 μg/ml). The action of beta-d-glucan was also compared with the properties of a well characterized commercial monomeric polyphenol - resveratrol (3,4′,5-trihydroxystilbene, the final concentration - 4 μg/ml). The two-way analysis variance showed that the differences in TBARS levels were depended on the type of tested drugs (p = 7.9 × 10⁻⁶). We observed a statistically increase of the level of biomarker of lipid peroxidation such as TBARS after 1 and 24 h incubation of plasma with haloperidol compared to the control samples (p < 0.01, p < 0.02, respectively). Amisulpride, contrary to haloperidol (after 1 and 24 h) did not cause plasma lipid peroxidation (p > 0.05). We showed that in the presence of beta-glucan, lipid peroxidation in plasma samples treated with haloperidol was significantly decreased. Moreover, we did not observe the synergistic action of beta-glucan and amisulpride on the inhibition of plasma lipid peroxidation. However, the beta-d-glucan was found to be more effective antioxidant, than the solution of pure resveratrol. The presented results indicate that beta-glucan seems to have distinctly protective effects against the impairment of plasma lipid molecules induced by haloperidol.     

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.     

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F₂-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F_2α (PGF_2α), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF_2α in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF_2α levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of “oxidative stress” or “oxidative damage” in vivo.     

Differential oxidative modification of proteins in MRL+/+ and MRL/lpr mice: Increased formation of lipid peroxidation-derived aldehyde-protein adducts may contribute to accelerated onset of autoimmune response

Even though reactive oxygen species (ROS) have been implicated in SLE pathogenesis, the contributory role of ROS, especially the consequences of oxidative modification of proteins by lipid peroxidation-derived aldehydes (LPDAs) such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) in eliciting an autoimmune response and disease pathogenesis remains largely unexplored. MRL/lpr mice, a widely used model for SLE, spontaneously develop a condition similar to human SLE, whereas MRL+/+ mice with the same MRL background, show much slower onset of SLE. To assess if the differences in the onset of SLE in the two substrains could partly be due to differential expression of LPDAs and to provide evidence for the role of LPDA-modified proteins in SLE pathogenesis, we determined the serum levels of MDA-/HNE-protein adducts, anti-MDA-/HNE-protein adduct antibodies, MDA-/HNE-protein adduct specific immune complexes, and various autoantibodies in 6-, 12- and 18-week old mice of both substrains. The results show age-related increases in the formation of MDA-/HNE-protein adducts, their corresponding antibodies and MDA-/HNE-specific immune complexes, but MRL/lpr mice showed greater and more accelerated response. Interestingly, a highly positive correlation between increased anti-MDA-/HNE-protein adduct antibodies and autoantibodies was observed. More importantly, we further observed that HNE-MSA caused significant inhibition in antinuclear antibodies (ANA) binding to nuclear antigens. These findings suggest that LPDA-modified proteins could be important sources of autoantibodies and CICs in these mice, and thus contribute to autoimmune disease pathogenesis. The observed differential responses to LPDAs in MRL/lpr and MRL+/+ mice may, in part, be responsible for accelerated and delayed onset of the disease, respectively.     

Protective role of arzanol against lipid peroxidation in biological systems

This study examines the protective effect of arzanol, a pyrone–phloroglucinol etherodimer from Helichrysum italicum subsp. microphyllum, against the oxidative modification of lipid components induced by Cu²⁺ ions in human low density lipoprotein (LDL) and by tert-butyl hydroperoxide (TBH) in cell membranes. LDL pre-treatment with arzanol significantly preserved lipoproteins from oxidative damage at 2 h of oxidation, and showed a remarkable protective effect on the reduction of polyunsaturated fatty acids and cholesterol levels, inhibiting the increase of oxidative products (conjugated dienes fatty acids hydroperoxides, 7β-hydroxycholesterol, and 7-ketocholesterol). Arzanol, at non-cytotoxic concentrations, exerted a noteworthy protection on TBH-induced oxidative damage in a line of fibroblasts derived from monkey kidney (Vero cells) and in human intestinal epithelial cells (Caco-2), decreasing, in both cell lines, the formation of oxidative products (hydroperoxides and 7-ketocholesterol) from the degradation of unsaturated fatty acids and cholesterol. The cellular uptake and transepithelial transport of the compound were also investigated in Caco-2 cell monolayers. Arzanol appeared to accumulate in Caco-2 epithelial cells. This phenol was able to pass through the intestinal Caco-2 monolayers, the apparent permeability coefficients (P_app) in the apical-to-basolateral and basolateral-to-apical direction at 2 h were 1.93 ± 0.36 × 10⁻⁵ and 2.20 ± 0.004 × 10⁻⁵ cm/s, respectively, suggesting a passive diffusion pathway. The results of the work qualify arzanol as a potent natural antioxidant with a protective effect against lipid oxidation in biological systems.     

Short-term and long-term vitamin C supplementation in humans dose-dependently increases the resistance of plasma to ex vivo lipid peroxidation

To assess the effects of short-term and long-term vitamin C supplementation in humans on plasma antioxidant status and resistance to oxidative stress, plasma was obtained from 20 individuals before and 2h after oral administration of 2g of vitamin C, or from eight subjects enrolled in a vitamin C depletion-repletion study using increasing daily doses of vitamin C from 30 to 2500 mg. Plasma concentrations of ascorbate, but not other physiological antioxidants, increased significantly after short-term supplementation, and increased progressively in the long-term study with increasing vitamin C doses of up to 1000 mg/day. Upon incubation of plasma with a free radical initiator, ascorbate concentrations were positively correlated with the lag phase preceding detectable lipid peroxidation. We conclude that vitamin C supplementation in humans dose-dependently increases plasma ascorbate concentrations and, thus, the resistance of plasma to lipid peroxidation ex vivo. Plasma and body saturation with vitamin C in humans appears desirable to maximize antioxidant protection and lower risk of oxidative damage.     

Oxidative stress in small-for-gestational age (SGA) term newborns and their mothers

This study used malondialdehyde (MDA) determination by HPLC and enzymatic assays for total serum peroxides and antioxidant capacity to evaluate oxidative stress in 47 healthy full-term small-for-gestational age (SGA) newborns vs 67 appropriate-for-gestational age (AGA) newborns. Blood samples were collected at delivery from umbilical cord artery and vein and from peripheral blood of the babies on the third day after birth. Blood samples of mothers were also collected and compared with blood of 29 normal non-pregnant women (NPW). Serum peroxide values were significantly higher in both groups of mothers than in NPW, decreasing towards the third day in AGA mothers, while persisting in SGA mothers. Antioxidant capacity of sera of both groups of mothers was lower than NPW. Both SGA mothers and babies had increased MDA at delivery, unlike AGA counterparts. MDA levels in umbilical vein were higher than in umbilical arteries, while immunohistochemistry revealed abundant presence of 4-hydroxynonenal (HNE)-protein adducts only in stroma of the SGA placenta. These results show that both mothers and babies are exposed to oxidative stress during and after delivery, which is more pronounced and persistent in the perinatal period of the SGA group, while lipid peroxidation in placenta could play a role in SGA pathophysiology.     

Arachidonic and docosahexaenoic acids reduce the growth of A549 human lung-tumor cells increasing lipid peroxidation and PPARs

Polyunsaturated fatty acids (PUFAs) play an important role in both induction and prevention of carcinogenic process. It is well known that several types of neoplastic cells show decreased total PUFA content, contributing to their resistance to chemotherapy and lipid peroxidation. In the light of this, human lung cancer A549 cells, with low PUFA content, were exposed to arachidonic or docosahexaenoic acid to investigate the effect of n-6 and n-3 PUFAs on growth and elucidate underlying mechanisms. The bulk of the results showed that both n-6 PUFAs and n-3 PUFAs decrease human lung-tumor cell growth in a concentration-dependent manner, inducing cell death mainly evident at 100microM concentration. The mechanism underlying the antiproliferative effect of n-6 and n-3 PUFAs appeared to be the same, involving changes in fatty acid composition of biomembranes, production of cytostatic aldehydes derived from lipid peroxidation and able to affect DNA-binding activity of AP-1, and induction of PPAR. From these results it may be hypothesized that n-6 PUFAs, like n-3 PUFAs, are able to inhibit tumor growth.     

Assessment of growth inhibition by aldehydic lipid peroxidation products and related aldehydes by Saccharomyces cerevisiae

The effect of pretreatment with aldehydes on the subsequent colony forming efficiency (CFE) of Saccharomyces cerevisiae was investigated. All 21 aldehydes tested inhibited CFE in a dose-dependent manner. The effective doses, however, differed markedly from 300 mM to 0·07 mM depending on the functional groups and chain length of the aldehydes. Amongst the nine representatives of n-alkanals, formaldehyde was the most potent inhibitor, reducing CFE to 50 per cent at a dose of 0·3 mM (IC₅₀). In the series of 2-trans-alkenals, acrolein was most effective with an IC₅₀ of 0·08 mM and amongst the 4-hydroxy 2-trans-alkenals, 4-hydroxynonenal was most effective with IC₅₀ of 0·07 mM . In general, effectiveness decreased in the order: 4-hydroxyalkenals > 2-alkenals ? n-alkenals. It is proposed that S. cerevisiae is a promising target cell to elucidate further the molecular mechanisms by which aldehydes, particularly the lipid peroxidation product 4-hydroxynonenal, inhibits cell proliferation.     

Plasma copper and lipid peroxidation in cigarette smokers

Plasma levels of copper and lipid peroxidation were evaluated in 14 smokers as compared to 14 nonsmokers. Plasma copper concentrations were higher in smokers than in nonsmokers (122.5 ± 19.15 vs. 101.5 ± 16.2 μg/dl, P < .01). Plasma lipoperoxidation, evaluated as fluorescent damage products of lipid peroxidation (FDPL), also was higher in smokers than in nonsmokers (20.35 ± 2.6 vs. 17.1 ± 2.95 units of relative fluorescence/ml, P < .01). A significant and positive correlation between the number of cigarettes smoked, expressed as pack years, and the levels of either FDPL (r = .61, P < .025) or copper (r = .55, P < .05) was found. Moreover, a significant and positive relationship between copper and FDPL values was observed in smokers (r = .64; P < .025), but not in nonsmokers. These data indicate that cigarette smoke-related plasma oxidant load may be partly due to enhanced levels of the prooxidant metal copper, potentially suggesting the supplementation of specific antioxidants (e.g., zinc) to counteract cigarette smoke-induced oxidative stress in smokers.     

Isoprostanes: novel bioactive products of lipid peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F₂-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F₂-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F₂-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.     

Exercise training protects against contraction-induced lipid peroxidation in the diaphragm

Endurance exercise training promotes a small but significant increase in antioxidant enzyme activity in the costal diaphragm (DIA) of rodents. It is unclear if these training-induced improvements in muscle antioxidant capacity are large enough to reduce oxidative stress during prolonged contractile activity. To test the hypothesis that training-related increases in DIA antioxidant capacity reduces contraction-induced lipid peroxidation, we exercise trained adult female Sprague-Dawley (n = 7) rats on a motor-driven treadmill for 12 weeks at approximately 75% maximal O2 consumption (90 min/day). Control animals (n = 8) remained sedentary during the same 12-week period. After training, DIA strips from animals in both experimental groups were excised and subjected to an in vitro fatigue contractile protocol in which the muscle was stimulated for 60 min at a frequency of 30 Hz, every 2 s, with a train duration of 330 m. Compared to the controls, endurance training resulted in an increase (P < 0.05) in diaphragmatic non-protein thiols and in the activity of the antioxidant enzyme superoxide dismutase. Following the contractile protocol, lipid peroxidation was significantly lower (P < 0.05) in the trained DIA compared to the controls. These data support the hypothesis that endurance exercise training-induced increases in DIA antioxidant capacity protect the muscle against contractile-related oxidative stress.     

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2-2. CeGSTP2-2 (the product of the gst-10 gene) has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4-HNE). Transgenic C. elegans strains were generated in which the 5'-flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with particularly high catalytic efficiency for 4-HNE. The localization of both transgenes was similar to that of native CeGSTP2-2. The 4-HNE-conjugating activity in worm lysates increased in the order: control相似文献