Total hydroxyoctadecadienoic acid as a marker for lipid peroxidation in vivo

An improved method for the measurement of lipid peroxidation in vivo has been recently developed, where total hydroxyoctadecadienoic acid (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomer, (Z, E)-HODE/(E, E)-HODE, could be also measured. In the present study, in order to examine the effect of continuous, slow flux of free radicals in vivo, a water-soluble radical generator was administered to rats and mice and the amounts of HODE and 8-isoprostane in plasma and liver were measured. It was found that the administration of free radical-generating azo compound increased the level of HODE and decreased the (Z, E)-HODE/(E, E)-HODE ratio in both plasma and liver. The level of HODE was much higher than 8-isoprostane.     

Bio-markers of lipid peroxidation in vivo: hydroxyoctadecadienoic acid and hydroxycholesterol

The biological role of lipid peroxidation products has continued to receive a great deal of attention not only for the elucidation of pathological mechanisms but also for their practical application to clinical use as bio-markers. In the last fifty years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. In this decade, F2-isoprostanes from arachidonates and neuroprostanes from docosahexanoates have been proposed as bio-markers. Although these markers are formed by a free radical-mediated oxidation, the yields from the parent lipids are minimal. Compared to these markers, hydroperoxy octadecadienoates (HPODE) from linoleates and oxysterols from cholesterols are yielded by much simpler mechanisms from more abundant parent lipids in vivo. Recently, the method in which both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as total hydroxyoctadecadienoic acid (tHODE) and 7-hydroxycholesterol (t7-OHCh), respectively, was proposed. The concentrations of tHODE and t7-OHCh determined by GC-MS analysis from physiological samples were much higher than that of 8-iso-prostagrandin F(2alpha). In addition to this advantage, hydrogen-donor activity of antioxidants in vivo could be determined by the isomeric-ratio of HODE (9- and 13-(Z,E)-HODE/9- and 13-(E,E)-HODE).     

Evaluation of the dietary effects of coenzyme Q in vivo by the oxidative stress marker, hydroxyoctadecadienoic acid and its stereoisomer ratio

Coenzyme Q (CoQ) is an endogenous enzyme cofactor that may provide protective benefits as an antioxidant. In this study, in order to determine whether the concentrations of CoQ(9) are associated with the oxidative status in vivo, the effects of dietary supplements of CoQ(9) on mice were evaluated by using a new biomarker, total hydroxyoctadecadienoic acid (tHODE). Biological samples were first reduced and then saponified to convert the various oxidation products of linoleates to tHODE. Subsequently, by using GC-MS analyses, we simultaneously determined the absolute concentration of tHODE; its stereoisomer ratio, 9- and 13-(Z,E)-HODE/9- and 13-(E,E)-HODE, which is a measure of the hydrogen donor capacity of antioxidants; and the concentration of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)). Remarkable decreases in tHODE and 8-iso-PGF(2alpha) levels were observed in the plasma, erythrocytes, liver, and brain of mice that were maintained for 1 month on an alpha-tocopherol (alphaT)-free (E-free) diet supplemented with ubiquinone-9 (Q(9); 0.04 wt.%) as compared to those of mice that were fed an E-free diet. The (Z,E/E,E) HODE ratio was increased in the plasma and erythrocytes of mice that were fed a Q(9)-fortified diet as compared to those that were fed an E-free diet. In particular, the (Z,E/E,E) HODE ratios in the plasma and brain were significantly correlated with the concentrations of ubiquinol-9 (Q(9)H(2)). Further, the liver and brain levels of tHODE and 8-iso-PGF(2alpha) were significantly correlated with the plasma and erythrocyte levels of tHODE and 8-iso-PGF(2alpha), respectively, and in some cases, also exhibited significant correlations with antioxidants. These results indicate that the plasma and erythrocyte levels of tHODE and its stereoisomeric ratio can be prominent biomarkers for the evaluation of the oxidative status and antioxidant capacity in vivo, including in the liver and brain, and that CoQ plays a major role in the in vivo antioxidant network.     

Vanadium distribution, lipid peroxidation and oxidative stress markers upon decavanadate in vivo administration

The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12h after metavanadate administration, whilst for decavanadate no effects were observed except 1h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases (-55%) mitochondrial CAT activity. At early times of exposure, 1 and 6h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.     

Detection of in vivo lipid peroxidation using the thiobarbituric acid assay for lipid hydroperoxides

Thiobarbituric acid (TBA) assays which have been modified for detection of lipid hydroperoxides appear to be useful for demonstration of in vivo lipid peroxidation. Since these methods require heating tissue membranes with the buffered TBA, there is a possibility of interference from the detection of autoxidation that occurs during heating. These studies were undertaken to investigate conditions which favor TBA color production from hydroperoxide while limiting autoxidation during the assay. An acetic acid-sodium acetate buffered (pH 3.6) TBA assay was used. Heating linoleic acid hydroperoxide with 50 microM ferric iron or under nitrogen nearly doubled color production compared to heating it with no added iron or under air. The lipid antioxidant butylated hydroxytoluene inhibited color production from fatty acid hydroperoxides. When tissue fractions, including liver and lung microsomes and lung whole membranes, were heated in the assay, color production was greater under air than under nitrogen and was much greater under oxygen. When liver microsomes from carbon tetrachloride-exposed rats were used, color was increased only when oxygen was present in the heating atmosphere. The results with tissue fractions appear to demonstrate autoxidation during color development rather than the presence of preformed hydroperoxides. Finally, it was found that color production from membrane fractions was dependent on the vitamin E content of the membranes. It appears that autoxidation during heating should be limited by heating under nitrogen and not by adding antioxidants, which inhibit color production from hydroperoxides. As the vitamin E effect demonstrates, antioxidant status must be considered, since a change in color production could result from a change in antioxidant content without the accumulation of lipid hydroperoxides.     

Determination of salicylate hydroxylation products as an in vivo oxidative stress marker

The in vivo measurement of highly reactive free radicals, such as the z.rad OH radical, is very difficult. New specific markers, which are based on the ability of z.rad OH to attack the benzene rings of aromatic molecules, are currently under investigation. The produced hydroxylated compounds can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids). The latter acid can be also produced by enzymatic pathways through the cytochrome P-450 system, while the former acid is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2, 3-DHBA, following oral administration of the drug acetyl salicylate, could be proposed for assessment of oxidative stress in vivo. In this paper, a sensitive method for the identification and quantification of hydroxylation products from the reaction of z. rad OH with salicylate in vivo is presented. It employs a high performance liquid chromatography and electrochemical detection system. A detection limit of < 1 pmol for the hydroxylation products has been achieved with linear response over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA dihydroxylated derivatives and salicylic acid and determined the ratios following administration of 1 g acetyl salicylate in 20 healthy subjects.     

Effect of docosahexaenoic acid intake on lipid peroxidation in diabetic rat retina under oxidative stress

Docosahexaenoic acid (DHA) plays an important role in visual function but has a highly oxidation-prone chemical structure. Therefore, we investigated how dietary DHA affects the generation of lipid peroxides in rat retina under oxidative stress in diabetes with/without vitamin E (VE) deficiency. Streptozotocin-induced (50 mg i.p./kg B.W.) diabetic Sprague-Dawley (SD) rats were assigned to four groups: (i) control/VE(+), (ii) DHA/VE(+), (iii) control/VE( - ) and (iv) DHA/VE( - ), and raised for 28 days. We then measured lipid peroxide levels in the retina, serum and liver. With a normal intake of VE, dietary DHA increased only the retinal level of thiobarbituric acid-reactive substances (TBARS) slightly. In contrast, in rats with VE deficiency, dietary DHA increased serum and liver lipid peroxide levels but not in the retina. These results suggest that dietary DHA does not necessarily promote lipid peroxidation in the retina even under high oxidative stress.     

Ophthalmic acid is a marker of oxidative stress in plants as in animals

Background

Ophthalmic acid (OPH), γ-glutamyl-L-2-aminobutyryl-glycine, a tripeptide analogue of glutathione (GSH), has recently captured considerable attention as a biomarker of oxidative stress in animals. The OPH and GSH biosynthesis, as well as some biochemical behaviors, are very similar. Here, we sought to investigate the presence of OPH in plants and its possible relationship with GSH, known to possess multiple functions in the plant development, growth and response to environmental changes.

Urinary aldehydes as indicators of lipid peroxidation in vivo

The excretion of malondialdehyde (MDA), lipophilic aldehydes and related carbonyl compounds in rat and human urine was investigated. MDA was found to be excreted mainly in the form of two adducts with lysine, indicating that its predominant reaction in vivo is with the lysine residues of proteins. Adducts with the phospholipid bases serine and ethanolamine and the nucleic acid bases guanine and deoxyguanosine also were found. Except for the adduct with deoxyguanosine (dG-MDA), the excretion of these compounds increased with peroxidative stress imposed in the form of vitamin E deficiency or the administration of iron or carbon tetrachloride. Marked differences in the concentration of dG-MDA in different tissues were correlated with their content of fatty acids having three or more double bonds, the putative source of MDA. Fourteen nonpolar and eleven polar lipophilic aldehydes and other carbonyl compounds were identified as their 2,4-diphenylhydrazine derivatives in rat urine. The excretion of five nonpolar and nine polar compounds was increased under conditions of peroxidative stress. The profile of lipophilic aldehydes obtained for human urine resembled that for rat urine. Except for a reported 4-hydroxynon-2-enal conjugate with mercapturic acid, the conjugated forms of the lipophilic aldehydes excreted in urine remain unidentified. Aldehyde excretion is influenced by numerous factors that affect the formation of lipid peroxides in vivo such as energy status, physical activity and environmental temperature, as well as by wide variations in the intake of peroxides in the diet. Consequently, urinalysis for aldehydic products of lipid peroxidation is an unreliable indicator of the general state of peroxidative stress in vivo.     

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.     

7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: Difference between cellular and plasma lipid peroxidation

Polyunsaturated fatty acids and their esters are known to be susceptible to free radical-mediated oxidation, whereas cholesterol is thought to be more resistant to oxidation. In fact, it has been observed that in the case of plasma lipid peroxidation, the amount of oxidation products of polyunsaturated fatty acids such as linoleic acid was higher than that of cholesterol. In contrast, during oxidative stress-induced cellular lipid peroxidation, oxidation products of cholesterol such as 7-hydroxycholesterol (7-OHCh) were detected in greater amounts than those of linoleates such as hydroxyoctadecadienoic acid (HODE). There are several forms of oxidation products of cholesterol and linoleates in vivo, namely, hydroperoxides, as well as the hydroxides of both the free and ester forms of cholesterol and linoleates. To evaluate these oxidation products, a method used to determine the lipid oxidation products after reduction and saponification was developed. With this method, several forms of oxidation products of cholesterol and linoleates are measured as total 7-OHCh (t7-OHCh) and total HODE (tHODE), respectively. During free radical-mediated lipid peroxidation in plasma, the amount of tHODE was 6.3-fold higher than that of t7-OHCh. In contrast, when Jurkat cells were exposed to free radicals, the increased amount of cellular t7-OHCh was 5.7-fold higher than that of tHODE. Higher levels of t7-OHCh than those of tHODE have also been observed in selenium-deficient Jurkat cells and glutamate-treated neuronal cells. These results suggest that, in contrast to plasma oxidation, cellular cholesterol is more susceptible to oxidation than cellular linoleates. Collectively, cholesterol oxidation products at the 7-position may be a biomarker of cellular lipid peroxidation.     

7-Hydroperoxycholesterol as a marker of oxidative stress in rat kidney induced by paraquat

The in vivo paraquat-induced oxidative stress in rat tissue was studied by analyzing cholesterol-derived hydroperoxide as an index of lipid peroxidation. Paraquat (10 mg/kg) was administered i.p. to rats. Rats were sacrificed and lung, liver, and kidney were collected 2, 24 h, and 5 d after paraquat injection. Lipids were extracted and analyzed by HPLC with post-column chemiluminescence. We found that two cholesterol-derived hydroperoxides, 7α-hydroperoxycholest-5-en-3β-ol (7α-OOH) and 7β-hydroperoxycholest-5-en-3β-ol (7β-OOH) were present in lungs of control animals (0.06 and 0.06 nmol/g, respectively), in livers (6.5 and 15.8 nmol/g, respectively) and in kidneys (3.7 and 8.9 nmol/g, respectively). In liver paraquat increased lipid peroxidation approximately by 60% over the levels of control animals only at 2 h after paraquat treatment. In kidney, augmented lipid peroxidation, 7α-OOH and 7β-OOH (by 70% and 147%, respectively) above levels was found at 2 h after paraquat treatment. Interestingly, these increase remained in kidney of rats 5 d after a single dose of paraquat. In contrast, cholesterol-derived hydroperoxides were not affected in lung of paraquat dosed rats. This is the first report on 7α-OOH and 7β-OOH accumulations in rat liver and kidney, and it seems to reflect greater oxidative stress in the pathology of kidney of rats treated with acute paraquat at low dose.     

7-Hydroperoxycholesterol as a marker of oxidative stress in rat kidney induced by paraquat

The in vivo paraquat-induced oxidative stress in rat tissue was studied by analyzing cholesterol-derived hydroperoxide as an index of lipid peroxidation. Paraquat (10 mg/kg) was administered i.p. to rats. Rats were sacrificed and lung, liver, and kidney were collected 2, 24 h, and 5 d after paraquat injection. Lipids were extracted and analyzed by HPLC with post-column chemiluminescence. We found that two cholesterol-derived hydroperoxides, 7alpha-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH) and 7beta-hydroperoxycholest-5-en-3beta-ol (7beta-OOH) were present in lungs of control animals (0.06 and 0.06 nmol/g, respectively), in livers (6.5 and 15.8 nmol/g, respectively) and in kidneys (3.7 and 8.9 nmol/g, respectively). In liver paraquat increased lipid peroxidation approximately by 60% over the levels of control animals only at 2 h after paraquat treatment. In kidney, augmented lipid peroxidation, 7alpha-OOH and 7beta-OOH (by 70% and 147%, respectively) above levels was found at 2 h after paraquat treatment. Interestingly, these increase remained in kidney of rats 5 d after a single dose of paraquat. In contrast, cholesterol-derived hydroperoxides were not affected in lung of paraquat dosed rats. This is the first report on 7alpha-OOH and 7beta-OOH accumulations in rat liver and kidney, and it seems to reflect greater oxidative stress in the pathology of kidney of rats treated with acute paraquat at low dose.     

Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients.

In vivo metabolism of salicylic acid produces two main hydroxylated derivatives (2,5- and 2,3-dihydroxybenzoic acid). The former can be produced by enzymatic pathways through the cytochrome P-450 system, while the latter is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2,3-dihydroxybenzoate, following oral administration of salicylate in its acetylated form (aspirin), has been proposed for assessment of oxidative stress. In this article we report plasma levels of 2,3- and 2,5-dihydroxybenzoates following the administration of 1 g aspirin and plasma levels of thiobarbituric acid-reactive material (TBARM) in well-controlled diabetic patients and in healthy subjects. 2,3-Dihydroxybenzoate levels were significantly higher (23%) in diabetic patients than in controls (63.4 +/- 20.1 versus 49.0 +/- 6.8 nM; p < .05). On the other hand, TBARM values were not significantly different between groups. These results suggest that the method is useful to reveal in vivo oxidative stress independently from the peroxidation of lipids, and they support the hypothesis that oxygen radicals are involved in the pathogenesis of chronic complications of diabetes.     

Breath ethane generation during clinical total body irradiation as a marker of oxygen-free-radical-mediated lipid peroxidation: A case study

Total body irradiation (TBI) is used therapeutically for treatment of leukemias and other malignancies of the hemopoietic system. Ionizing radiation produces oxygen free radicals that contribute to cytotoxicity. Breath collected from one patient undergoing therapeutic TBI showed measurable changes in levels of ethane during treatment. Breath ethane is a marker of lipid peroxidation of n-3 fatty acids. The TBI treatment involved 4 days of irradiation. The largest changes in breath ethane occured on Day 2. The increased levels of breath ethane on Day 2 were correlated to clinical manifestations of toxicity. The correlation of the onset of gastrointestinal side effects with higher levels of breath ethane suggests that breath ethane may be a clinically useful measure of the toxicity of various TBI fractionation treatment protocols currently in use at different medical centers. The levels of breath ehtane on the other days of treatment were lower, suggesting that the oxidative-antioxidative balance of the patient may be important in protection against free radical mediated injury. These results for a single patient suggest that breath ethane may be a promising approach to elucidate the role of antioxidants in clinical TBI and should be extended for verification to a larger volunteer patient population.     

Method development and validation for monitoring in vivo oxidative stress: evaluation of lipid peroxidation and fat-soluble vitamin status by HPLC in rat plasma

Monitoring in vivo oxidative stress implicates the evaluation of damage and defence parameters by well-established, validated methods. We report two optimized and validated HPLC methods for measurement of malondialdehyde (MDA) and fat-soluble vitamins in rat plasma. For the MDA method, optimization experiments of the thiobarbituric acid test resulted in the addition of 1% butylhydroxytoluene to the reaction mixture and in a heating time reduction to 40 min, ensuring inhibition of further lipid peroxidation during the test. Validation experiments showed good linearity, precision and recovery. The use of HPLC with coulometric array detection technology permits simultaneous and sensitive analysis of different fat-soluble vitamins and related compounds (tocopherols, retinoids, carotenoids and coenzyme Q10), which are identified by both retention time and electrochemical characteristics. Furthermore, this method is extended to the analysis of coenzyme Q9, the predominant homologue in rats. Validation experiments with rat plasma gave good results.     

Exocyclic DNA adducts as oxidative stress markers in colon carcinogenesis: potential role of lipid peroxidation,dietary fat and antioxidants

Molecular pathways to colorectal cancer involve multiple genetic changes, whereby extensive oxyradical damage causes mutations in cancer-related genes and leads to a cycle of cell death and regeneration. Besides direct oxidative DNA-damage, reactive oxygen and nitrogen species can induce etheno (epsilon)-DNA adducts mainly via trans-4-hydroxy-2-nonenal, generated as the major aldehyde by lipid peroxidation (LPO) of omega-6 PUFAs. Patients with familial adenomatous polyposis (FAP) develop multiple colorectal adenomas. In affected tissues increased LPO could be triggered due to increased arachidonic acid metabolism as a result of elevated cyclooxygenases. Our studies demonstrated an increased epsilon-DNA adduct level in affected colon epithelia of FAP patients. Epsilon-DNA adducts are promutagenic and can cause genomic instability that drives colorectal adenoma to malignancy. We have further investigated the potential chemopreventive properties of olive oil and its polyphenolic components. 'Mediterranean diet', of which olive oil is a major fatty acid source, has protective effects against human breast and colorectal cancers. Olive oil extracts and the newly identified lignan fractions showed high antioxidant capacity in vitro. As epsilon-DNA adducts are biomarkers for oxidative stress and LPO induced DNA damage, they can verify the efficacy of newly identified antioxidants, e.g. from olive oil, as chemopreventive agents against colon carcinogenesis.