Free F2-isoprostanes Research Articles

Mitochondria-targeted antioxidant supplementation improves 8\u2009km time trial performance in middle-aged trained male cyclists

BackgroundExercise increases skeletal muscle reactive oxygen species (ROS) production, which may contribute to the onset of muscular fatigue and impair athletic performance. Mitochondria-targeted antioxidants such as MitoQ, which contains a ubiquinone moiety and is targeted to mitochondria through the addition of a lipophilic triphenylphosphonium cation, are becoming popular amongst active individuals as they are designed to accumulate within mitochondria and may provide targeted protection against exercise-induced oxidative stress. However, the effect of MitoQ supplementation on cycling performance is currently unknown. Here, we investigate whether MitoQ supplementation can improve cycling performance measured as time to complete an 8 km time trial.MethodIn a randomized, double-blind, placebo-controlled crossover study, 19 middle-aged (age: 44 ± 4 years) recreationally trained (VO2peak: 58.5 ± 6.2 ml·kg− 1·min− 1, distance cycled per week during 6 months prior to study enrollment: 158.3 ± 58.4 km) male cyclists completed 45 min cycling at 70% VO2peak followed by an 8 km time trial after 28 days of supplementation with MitoQ (20 mg·day− 1) and a placebo. Free F2-isoprostanes were measured in plasma samples collected at rest, after 45 min cycling at 70% VO2peak and after completion of the time trial. Respiratory gases and measures of rating of perceived exertion (RPE) were also collected.ResultsMean completion time for the time trial was 1.3% faster with MitoQ (12.91 ± 0.94 min) compared to placebo (13.09 ± 0.95 min, p = 0.04, 95% CI [0.05, 2.64], d = 0.2). There was no difference in RPE during the time trial between conditions (p = 0.82) despite there being a 4.4% increase in average power output during the time trial following MitoQ supplementation compared to placebo (placebo; 270 ± 51 W, MitoQ; 280 ± 53 W, p = 0.04, 95% CI [0.49, 8.22], d = 0.2). Plasma F2-isoprostanes were lower on completion of the time trial following MitoQ supplementation (35.89 ± 13.6 pg·ml− 1) compared to placebo (44.7 ± 16.9 pg·ml− 1p = 0.03).ConclusionThese data suggest that MitoQ supplementation may be an effective nutritional strategy to attenuate exercise-induced increases in oxidative damage to lipids and improve cycling performance.

Perioperative intravenous acetaminophen attenuates lipid peroxidation in adults undergoing cardiopulmonary bypass: a randomized clinical trial.

BackgroundCardiopulmonary bypass (CPB) lyses erythrocytes and induces lipid peroxidation, indicated by increasing plasma concentrations of free hemoglobin, F2-isoprostanes, and isofurans. Acetaminophen attenuates hemeprotein-mediated lipid peroxidation, reduces plasma and urine concentrations of F2-isoprostanes, and preserves kidney function in an animal model of rhabdomyolysis. Acetaminophen also attenuates plasma concentrations of isofurans in children undergoing CPB. The effect of acetaminophen on lipid peroxidation in adults has not been studied. This was a pilot study designed to test the hypothesis that acetaminophen attenuates lipid peroxidation in adults undergoing CPB and to generate data for a clinical trial aimed to reduce acute kidney injury following cardiac surgery.Methods and ResultsIn a prospective double-blind placebo-controlled clinical trial, sixty adult patients were randomized to receive intravenous acetaminophen or placebo starting prior to initiation of CPB and for every 6 hours for 4 doses. Acetaminophen concentrations measured 30 min into CPB and post-CPB were 11.9±0.6 μg/mL (78.9±3.9 μM) and 8.7±0.3 μg/mL (57.6±2.0 μM), respectively. Plasma free hemoglobin increased more than 15-fold during CPB, and haptoglobin decreased 73%, indicating hemolysis. Plasma and urinary markers of lipid peroxidation also increased during CPB but returned to baseline by the first postoperative day. Acetaminophen reduced plasma isofuran concentrations over the duration of the study (P = 0.05), and the intraoperative plasma isofuran concentrations that corresponded to peak hemolysis were attenuated in those subjects randomized to acetaminophen (P = 0.03). Perioperative acetaminophen did not affect plasma concentrations of F2-isoprostanes or urinary markers of lipid peroxidation.ConclusionsIntravenous acetaminophen attenuates the increase in intraoperative plasma isofuran concentrations that occurs during CPB, while urinary markers were unaffected.Trial RegistrationClinicalTrials.gov NCT01366976

Aerobic training reduces systemic oxidative stress in young women with elevated levels of F2-isoprostanes

The purpose of this trial was to determine whether exercise without weight loss would reduce F2-isoprostanes in previously sedentary young women. Participants (N=319) were randomized to four months of 150min/week aerobic exercise or a control group. Plasma free F2-isoprostanes were measured by gas chromatography–mass spectrometry. Other assessments included fitness and % body fat. Intervention groups were compared with stratification by quartiles of baseline plasma F2-isoprostane. This stratified comparison was linearly adjusted for baseline plasma F2-isoprostane and we also used baseline plasma F2-isoprostane as a propensity score to balance the intervention. Training resulted in significant improvements in aerobic fitness (0.88 METs) and body fat percent (1%) in exercisers compared to controls (p<0.001). The exercise group had significantly higher mean baseline plasma F2-isoprostanes (79.1 vs 67.9pg/mL) than the control group in the highest quartile of baseline plasma F2-isoprostanes. Within this highest quartile, exercise led to a greater decline in plasma F2-isoprostanes (−20.2±2.5pg/mL) than control (−7.4±2.5pg/mL); with adjustment for baseline plasma F2-isoprostanes and in the balanced groups, this difference was reduced but remained significant. Four months of exercise training resulted in significant reductions of systemic oxidative stress only among previously sedentary young women who were in the highest quartile of plasma F2-isoprostanes at baseline (≥57pg/mL). Our findings indicate that the benefits of aerobic exercise in reducing systemic oxidative stress may be limited to those who present higher baseline levels of plasma F2-isoprostanes.

Concentration of isoprostanes as a marker reflecting oxidative status in moderate dyslipidemia

Dyslipidemia and oxidative stress are considered one of the major causes which lead to many diseases, especially atherosclerosis development, cardiovascular disorders and aggravate risk for cancers, aging and others. Lipid hydroperoxides are the initial products when lipids are damaged by free radicals and in plasma HDL is their major carrier. F2-IsoP are stable compounds and specific products of lipid peroxidation only in phospholipids in vivo through nonenzymatic free-radical mechanisms. Also, they are found in detectable quantities esterified in all biological tissues and in free form in all normal biological fluids. Quantifying of them is considered as accurate way to measure oxidative stress in vivo and could be used as an indicator of lipid peroxidation which process is a risk factor for atherosclerosis and other systemic diseases. Our study was performed in the group of young people (n=10, age 19-30) with moderate dyslipidemia and the reference group. In both groups we have investigated serum lipids (TG, TC, LDL-C, HDL-C, nonHDL-C), lipoproteins (apoAI, apoB) and lipid (TC/HDL-C, TG/HDL-C, LDL-C/HDL-C) and lipoprotein (HDL-C/apoAI) ratios and oxidative status by measuring free and total (free and esterified) F2-isoprostanes (F2-IsoP). According to the available data reports and taking into account results obtained in our study we concluded that even mild lipid and lipoprotein abnormalities which are often present in population might by connected with elevated oxidative status which is sensitively reflected by F2-IsoP concentration. Thus measurement of F2-IsoP seems to be very helpful in recognition of early stage of oxidative stress however further studies are required.

Biomarkers of oxidative damage in cigarette smokers: Which biomarkers might reflect acute versus chronic oxidative stress?

Cigarette smoking predisposes to the development of multiple diseases involving oxidative damage. We measured a range of oxidative damage biomarkers to understand which differ between smokers and nonsmokers and if the levels of these biomarkers change further during the act of smoking itself. Despite overnight abstinence from smoking, smokers had higher levels of plasma total and esterified F2-isoprostanes, hydroxyeicosatetraenoic acid products (HETEs), F4-neuroprostanes, 7-ketocholesterol, and 24- and 27-hydroxycholesterol. Levels of urinary F2-isoprostanes, HETEs, and 8-hydroxy-2′-deoxyguanosine were also increased compared with age-matched nonsmokers. Several biomarkers (plasma free F2-isoprostanes, allantoin, and 7β-hydroxycholesterol and urinary F2-isoprostane metabolites) were not elevated. The smokers were then asked to smoke a cigarette; this acute smoking elevated plasma and urinary F2-isoprostanes, plasma allantoin, and certain cholesterol oxidation products compared to presmoking levels, but not plasma HETEs or urinary 8-hydroxy-2′-deoxyguanosine. Smokers showed differences in plasma fatty acid composition. Our findings confirm that certain oxidative damage biomarkers are elevated in smokers even after a period of abstinence from smoking, whereas these plus some others are elevated after acute smoking. Thus, different biomarkers do not measure identical aspects of oxidative stress.

Analytical and Biological Variation of F2-Isoprostanes during the Menstrual Cycle

F2-isoprostanes have been advocated as a specific and reliable marker of oxidative damage (1) despite a lack of data on their biological and analytical variation. Concurrent with a longitudinal study of oxidative stress during the menstrual cycle [the BioCycle Study(2)], we have characterized the components of biological and analytical variation of free plasma F2-isoprostanes in the context of the menstrual cycle. We describe several of the key characteristics of F2-isoprostanes as a biomarker. Samples were collected from 9 healthy volunteers who met the major inclusion criteria of being a premenopausal woman 18–44 years of age and having regular menstrual cycles. Major exclusion criteria included use of hormonal preparations or contraceptive devices, pregnancy, a history of gynecologic abnormalities, recent infectious disease, routine intake of prescription and over-the-counter medications (including vitamin and mineral supplements), and a history of certain chronic diseases or conditions. Fasting blood samples were collected between 0700 and 0830 on days 2, 7, 12, 13, 14, 18, 22, and 27 of a single menstrual cycle. Collection and handling protocols were designed to minimize preanalytical variation. Samples were frozen at −80 °C and then analyzed, in duplicate, as a single set of consecutive samples by GC-MS (1) at the Molecular Epidemiology and Biomarker Research Laboratory at …

Effects of 50 Hz electromagnetic fields on rat cortical synaptosomes

Nerve cells are very responsive to weak pulsed electromagnetic fields (EMFs). Such non-ionizing radiation, with frequencies of 0-300 Hz and 0.1-100 mT, can affect several cellular activities, with unusual dose-response characteristics. The present study examined the effect of a 2-h exposure of synaptosomes on a system generating a peak magnetic field of 2 mT. We evaluated the changes of the synaptosomal mitochondrial respiration rate and ATP production, membrane potential, intrasynaptosomal Ca2+ concentration, and the release of free iron and F2-isoprostanes. O2 consumption and ATP production remained unchanged in exposed synaptosomes. The intrasynaptosomal Ca2+ concentration decreased slowly and no depolarization of the synaptosomal membrane was detected. Finally, the release of free iron and F2-isoprostanes by synaptosomal suspensions also remained unchanged after EMF exposure. These results indicate that the physiological behavior of cortical synaptosomes was unaffected by weak pulsed EMFs.

Isoprostanes

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo via a nonenzymatic mechanism involving the free radical-initiated peroxidation of arachidonic acid. This article summarizes our current knowledge of these compounds. Herein, a historical account of their discovery and the mechanism of their formation are described. A specific class of IsoPs, the F2-IsoPs, are stable, robust molecules that can be measured as indices of endogenous oxidant stress. The utility of these molecules as biomarkers and methods by which these compounds can be quantified are discussed. In addition to the F2-IsoPs, isoprostanes with other prostane ring structures as well as oxidation products with furan and dioxolane rings can be generated from arachidonic acid. And, in more recent years, isoprostane-like compounds have been shown to be formed from polyunsaturated fatty acids including eicosapentaenoic acid [C20:5, omega-3], docosahexaenoic acid [C22:6, omega-3], and adrenic acid [C22:4, omega-6]. These findings will be summarized as well.

HDL, lipid peroxidation, and atherosclerosis

Genetic, clinical, and pharmacological studies implicate elevated levels of LDL in the pathogenesis of atherosclerotic vascular disease, the leading cause of death in industrialized societies (1). Paradoxically, native LDL fails to exert potentially atherogenic effects in vitro, suggesting that it must be modified to promote vascular disease. Indeed, many lines of evidence support the LDL oxidation hypothesis, which suggests that oxidative damage to LDL is one important mechanism for rendering lipoproteins atherogenic (as reviewed in Refs. 2, 3).

The emerging roles of PAF acetylhydrolase

Platelet-activating factor (PAF), a phospholipid autacoid with potent effects throughout the innate immune system, is selectively degraded by two small families of PAF acetylhydrolases (PAF-AHs). These Ca2+-independent phospholipases A2 display remarkable specificity for the length of the sn-2 residue, but this selectivity is lost as the residue gains oxygen functions. Two of the PAF-AHs therefore are specific oxidized phospholipid phospholipases that reduce inflammation, but also remove oxidatively truncated phospholipids that induce apoptosis. The roles of these enzymes are manifold, and their separate and combined functions are now being addressed in model systems and clinical studies.

Plasma Esterified F2-Isoprostanes and Oxidative Stress in Newborns: Role of Nonprotein-Bound Iron

Nonprotein-bound iron (NPBI) and F2-isoprostanes, reliable markers of oxidative stress, are increased in plasma of newborns and inversely correlated to the gestational age. Because NPBI represents a pro-oxidant stimulus in plasma, we test the hypothesis that the entity of lipid peroxidation is related with NPBI concentrations. Plasma levels of free, esterified, and total F2-isoprostanes were investigated in relation to NPBI levels in 59 newborns and 16 healthy adults. The pro-oxidant role of iron was ascertained in vitro, by measuring all the forms of F2-isoprostanes after incubation with ammonium iron sulfate. Significant positive correlations were found between NPBI and total as well as esterified F2-isoprostanes in plasma of the newborns. The addition of ammonium iron sulfate induced a marked increase in all the forms of F2-isoprostanes after 2 hours of incubation. The higher NPBI concentration, the higher F2-isoprostanes levels. An increase NPBI dose dependent in total F2-isoprostanes formation was observed in dialyzed low density lipoprotein from adult plasma. The results clearly show that once NPBI is generated, whatever its source, it is capable of inducing oxidative stress. NPBI-induced oxidative stress may contribute to the morbidity in preterm infants that are particularly susceptible to free radical damage.

Serum Concentrations of F2-Isoprostanes and 4-Hydroxynonenal in Hemodialysis Patients in Relation to Inflammation and Renal Anemia

BACKGROUND: Patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD) are apparently exposed to enhanced oxidative stress and to inflammation. It was the aim of this study to characterize the state of systemic oxidative stress of ESRD patients before and following HD using highly specific biomarkers, F(2)-isoprostanes and 4-hydroxynonenal (HNE). Furthermore the question should be answered, if there are associations between inflammation and systemic oxidative stress and/or between systemic oxidative stress and renal anemia, which is more or less typical for HD patients. PATIENTS AND METHODS: Concentrations of F(2)-isoprostanes, HNE, C-reactive protein (CRP) as marker of inflammation, and hemoglobin were measured in serum samples of patients with ESRD before and after HD and of healthy control persons for comparison. Total (esterified plus free) F(2)-isoprostanes were quantified by highly sensitive gas chromatography/mass spectrometry technique, HNE by thin layer chromatography and HPLC/UV detection, CRP by immunoturbidimetry and hemoglobin by clinico-chemical routine assay. RESULTS: 1. HD patients showed significantly higher serum concentrations of F(2)-isoprostanes and HNE than healthy human control subjects. 2. Total (esterified plus free) F(2)-isoprostane levels before HD were not significantly different from those after HD, whereas HNE levels were significantly decreased in patients after HD. 3. F(2)-isoprostane concentrations in HD patients correlated with the levels of CRP, whereas HNE concentrations inversely correlated with the content of hemoglobin. CONCLUSION: Both, F(2)-isoprostanes and HNE serum concentrations are useful oxidative stress parameters in ESRD patients undergoing HD. Whereas HNE strongly correlates with the severity of renal anemia, leading to left heart insufficiency, F(2)-isoprostanes (sum of free plus esterified) highly correlate with the degree of inflammation.

BODY MASS INDEX INDEPENDENTLY PREDICTS OXIDATIVE STRESS AND INFLAMMATION IN STAGE II THROUGH IV CHRONIC KIDNEY DISEASE.

Oxidative stress and inflammation are highly prevalent in chronic kidney disease (CKD) and may explain the increased atherosclerotic burden in this population. Obesity is independently associated with increasing oxidative stress and inflammation in the general population. The purpose of this study is to investigate whether obesity, as assessed by body mass index (BMI), can predict oxidative stress and inflammation in patients with CKD. A cross-sectional observational study was conducted on 200 stage II through IV CKD patients. Demographics, cardiovascular disease history, markers of oxidative stress (free F2-isoprostanes, a marker of lipid peroxidation, measured by GC/MS) and inflammation (C-reactive protein [CRP]), and BMI were measured in all patients. Using Spearman rank correlations, BMI was significantly correlated with F2-isoprostanes (r = .271, p

Angiotensin II Induces Interleukin-6 in Humans Through a Mineralocorticoid Receptor–Dependent Mechanism

This study tested the hypothesis that angiotensin promotes oxidative stress and inflammation in humans via aldosterone and the mineralocorticoid receptor. We measured the effect of intravenous aldosterone (0.7 mug/kg per hour for 10 hours followed by 0.9 mug/kg per hour for 4 hours) and vehicle in a randomized, double-blind crossover study in 11 sodium-restricted normotensive subjects. Aldosterone increased interleukin (IL)-6 (from 4.7+/-4.9 to 9.4+/-7.1 pg/mL; F=4.94; P=0.04) but did not affect blood pressure, serum potassium, or high-sensitivity C-reactive protein. We next conducted a randomized, double-blind, placebo-controlled, crossover study to measure the effect of 3-hour infusion of angiotensin II (2 ng/kg per minute) and norepinephrine (30 ng/kg per minute) on separate days after 2 weeks of placebo or spironolactone (50 mg per day) in 14 salt-replete normotensive subjects. Angiotensin II increased blood pressure (increase in systolic pressure: 13.7+/-7.5 and 15.2+/-9.4 mm Hg during placebo and spironolactone, respectively; P<0.001 for angiotensin II) and decreased renal plasma flow (-202+/-73 and -167+/-112 mL/min/1.73 kg/m(2); P<0.001 for angiotensin II effect) similarly during placebo and spironolactone. Spironolactone enhanced the aldosterone response to angiotensin II (increase of 17.0+/-10.6 versus 9.0+/-5.7 ng/dL; P=0.002). Angiotensin II transiently increased free plasma F(2)-isoprostanes similarly during placebo and spironolactone. Angiotensin II increased serum IL-6 concentrations during placebo (from 1.8+/-1.1 to 2.4+/-1.4 pg/mL; F=4.5; P=0.04) but spironolactone prevented this effect (F=6.4; P=0.03 for spironolactone effect). Norepinephrine increased blood pressure and F(2)-isoprostanes but not aldosterone or IL-6. Aldosterone increases IL-6 in humans. These data suggest that angiotensin II induces IL-6 through a mineralocorticoid receptor-dependent mechanism in humans. In contrast, angiotensin II-induced oxidative stress, as measured by F(2)-isoprostanes, is mineralocorticoid receptor independent and may be pressor dependent.

Inhibitory effect of melatonin on diquat‐induced lipid peroxidation in vivo as assessed by the measurement of F2‐isoprostanes

Melatonin is a powerful antioxidant and free radical scavenger. A large body of in vivo and in vitro evidence shows that melatonin effectively inhibits membrane lipid peroxidation; this damage was based on the measurement of malondialdehyde and/or 4-hydroxynonenal levels. In the current study, for the first time using a more sensitive and specific biomarker, i.e. F2-isoprostanes, we investigate the effect of melatonin on diquat-induced lipid peroxidation in Fischer 344 rats. When diquat (40 mg/kg body weight) was intraperitoneally injected into rats, the levels of liver F2-isoprostanes were significantly increased at 1, 3, and 6 hr while plasma free F2-isoprostanes concentrations were augmented at 3, 6, and 12 hr after administration of the toxin. In addition, the plasma alanine aminotransferase activity level was measured as a parameter of hepatoxicity; the activity of this enzyme was augmented at 3, 6, and 12 hr after diquat administration when compared with levels of this constituent in untreated control rats. Pretreatment with melatonin (20 mg/kg) 30 min before diquat administration resulted in a significant reduction in both tissue and plasma F2-isoprostanes levels, and plasma alanine aminotransferase activity. These findings, using a sensitive and specific index of lipid peroxidation, show that the hepatoxicity of diquat, at least partially, is a consequence of reactive oxygen species-induced lipid damage. Melatonin's protective effects likely relate to its direct free radical scavenging ability and/or due to other antioxidative processes induced by the indole.

Release of Free F2-isoprostanes from Esterified Phospholipids Is Catalyzed by Intracellular and Plasma Platelet-activating Factor Acetylhydrolases

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Transgenic Mice Overexpressing Glutathione Peroxidase 4 Are Protected against Oxidative Stress-induced Apoptosis

Glutathione peroxidase 4 (Gpx4) is uniquely involved in the detoxification of oxidative damage to membrane lipids. Our previous studies showed that Gpx4 is essential for mouse survival and that Gpx4 deficiency makes cells vulnerable to oxidative injury. In the present study, we generated two lines of transgenic mice overexpressing Gpx4 (Tg(GPX4) mice) using a genomic clone containing the human GPX4 gene. Both lines of Tg-(GPX4) mice, Tg5 and Tg6, had elevated levels of Gpx4 (mRNA and protein) in all tissues investigated, and overexpression of Gpx4 did not cause alterations in activities of glutathione peroxidase 1, catalase, Cu/Zn superoxide dismutase, and manganese superoxide dismutase. The human GPX4 transgene rescued the lethal phenotype of null mutation of the mouse Gpx4 gene, indicating that the transgene can replace the essential role of mouse Gpx4 in mouse development. Cell death induced by t-butylhydroperoxide and diquat was significantly less in murine embryonic fibroblasts from Tg(GPX4) mice compared with wild type mice. Liver damage and lipid peroxidation induced by diquat were reduced significantly in Tg(GPX4) mice. In addition, diquat-induced apoptosis was decreased in Tg(GPX4) mice, as evidenced by attenuated caspase-3 activation and reduced cytochrome c release from mitochondria. These data demonstrate that Gpx4 plays a role in vivo in the mechanism of apoptosis induced by oxidative stress that most likely occurs through oxidative damage to mitochondrial phospholipids such as cardiolipin.

Amelioration of myoglobinuric renal damage in rats by chronic exposure to flavonol-rich red wine.

Myoglobinuric acute renal failure causes increased oxidative stress. Since ethanol upregulates renal antioxidant enzymes and wine polyphenols behave as antioxidants, we tested the hypothesis that red wine components would ameliorate the renal damage caused by rhabdomyolysis. Adult rats received water (control), alcohol-free red wine, ethanol 12.5% (v/v) or red wine for 10 weeks. Rhabdomyolysis was induced by glycerol injection (50%, 10 ml/kg, i.m.), and urine and blood samples were collected 6 h later to measure renal function parameters, creatine kinase (CK) activity, free F(2)-isoprostanes and total antioxidant capacity. Kidneys were then harvested for morphological studies and determinations of lipid peroxidation, protein carbonylation, (Na + K)-ATPase and antioxidant enzyme activities. In the control group, myoglobinuria was associated with a 68% decrease in creatinine clearance and increases in plasma creatinine and blood urea nitrogen of 3.2 and 1.8 times above baseline, respectively. Controls also showed increases in plasma free F(2)-isoprostanes levels and CK activity, together with enhanced renal expression of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase, as well as increased production of malondialdehyde and carbonyls. Rhabdomolysis reduced renal (Na + K)-ATPase activity and this reduction was associated with a 5-fold increase in fractional sodium excretion as well as morphological damage to the kidney. These changes were significantly attenuated by pretreatment with chronic red wine exposure prior to glycerol injection. A less marked degree of functional and biochemical protection was also observed in response to the administration of alcohol-free red wine and ethanol. The present data suggest that red wine protects against functional, biochemical and morphological damage caused by rhabdomyolysis in the rat, and this protection may be due to the synergistic effects of ethanol and non-alcoholic red wine components.

Enzyme immunoassays for 15-F2T isoprostane-M, an urinary biomarker for oxidant stress.

Isoprostane assays have significant potential for use as molecular biomarkers of human oxidant stress (Morrow et al. 1999, Lawson et al. 1999). Recent studies indicate that: (a) significant amounts of F2 isoprostanes derived from systemic oxidant stress are metabolized before excretion, (b) a significant quantity of free F2 isoprostanes are produced by the kidney, so free isoprostanes detected in urine may not accurately reflect systemic oxidant stress and (c) 2,3-dinor-5,6-dihydro-15-F2tIsoprostane (F2-IsoP-M, IsoP-M), a major urinary metabolite of systemic 8-iso-PG F2Œ, has recently been identified (Figure 1). Urinary F2-IsoP-M may, therefore, prove to be an excellent marker for systemic oxidant stress. Our goal is to develop a sensitive and specific immunoassay for F2-IsoP-M, which should provide a simple non-invasive method for routine quantitative assessment of human oxidant stress.

Increase in plasma esterified F2-isoprostanes following intravenous iron infusion in patients on hemodialysis

In epoetin-treated dialysis patients, currently iron is administered by the intravenous route to maintain optimum erythropoiesis. However, rapid infusion of iron in excess of transferrin binding capacity can lead to the availability of unbound iron that can theoretically catalyze peroxidation of lipids, such as low-density lipoprotein (LDL), which when oxidatively modified is proinflammatory and promotes atherogenesis. To address this issue, our study used one of the most specific measures of lipid peroxidation available, namely gas chromatography/mass spectometry (GC/MS) analysis of F2-isoprostanes. Using a prospective design, blood samples were collected 15 minutes before (pre) and 30 minutes after (post) a one-hour infusion of 700 mg bolus of intravenous iron in 22 adult home-hemodialysis patients on a non-hemodialysis day. With iron-dextran infusion, serum iron markedly increased (mean +/- SE, 42 +/- 4 vs. 311 +/- 92 microg/dL, P < 0.0001) and exceeded the transferrin saturation of 100% in 22 out of 22 patients (pre 23 +/- 3 vs. post 165 +/- 8%, P < 0.0001). Plasma concentrations of free F2-isoprostanes did not change significantly following infusion of iron (pre 40 +/- 5 vs. post 39 +/- 6 pg/mL). However, levels of F2-isoprostanes esterified in plasma lipoproteins increased significantly in the postinfusion samples (pre 199 +/- 19 vs. post 233 +/- 25 pg/mL, P < 0.004). Pre-infusion levels of serum iron correlated directly with pre-infusion levels of esterified F2-isoprostanes (r = 0.56, P = 0.008), which persisted in the postinfusion period (r = 0.43, P = 0.04). However, there was no correlation between esterified F2-isoprostanes and serum ferritin levels. In the last four patients in whom blood samples were collected five hours after the intravenous iron infusion, there were further increases in esterified F2-isoprostanes that very closely correlated with postinfusion serum iron levels (r = 0.99, P = 0.013). In a control study, the in vitro addition of iron dextran to blood samples did not increase free or esterified F2-isoprostanes, suggesting that the increase in esterified F2-isoprostanes seen in vivo after iron infusion in patients is not due to a procedural artifact. Collectively our data suggest that high levels of serum iron appearing soon after a large bolus of iron infusion is associated with significant, albeit modest, increases in levels of F2-isoprostanes esterified in plasma lipoproteins that tended to increase with time. Although it is uncertain whether this degree of lipid peroxidation may have deleterious effects, it may be sagacious to explore whether this can be prevented by slow infusion of frequent smaller doses of iron and, if necessary, along with administration of antioxidants.