Mitochondrial Isolevuglandins Contribute to Vascular Oxidative Stress and Mitochondria-Targeted Scavenger of Isolevuglandins Reduces Mitochondrial Dysfunction and Hypertension.

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II-infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II-infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine reduces atherosclerosis in hypercholesterolemic Ldlr-/- mice.

Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr-/- mice, a model of FH. Compared to hypercholesterolemic Ldlr-/- mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr-/- mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.

Intraoperative cerebral oxygenation, oxidative injury, and delirium following cardiac surgery.

BACKGROUND: Delirium affects 20-30% of patients after cardiac surgery and is associated with increased mortality and persistent cognitive decline. Hyperoxic reperfusion of ischemic tissues increases oxidative injury, but oxygen administration remains high during cardiac surgery. We tested the hypothesis that intraoperative hyperoxic cerebral reperfusion is associated with increased postoperative delirium and that oxidative injury mediates this association. METHODS: We prospectively measured cerebral oxygenation with bilateral oximetry monitors in 310 cardiac surgery patients, quantified intraoperative hyperoxic cerebral reperfusion by measuring the magnitude of cerebral oxygenation above baseline after any ischemic event, and assessed patients for delirium twice daily in the ICU following surgery using the confusion assessment method for ICU (CAM-ICU). We examined the association between hyperoxic cerebral reperfusion and postoperative delirium, adjusted for the extent of cerebral hypoxia, the extent of cerebral hyperoxia prior to any ischemia, and additional potential confounders and risk factors for delirium. To assess oxidative injury mediation, we examined the association between hyperoxic cerebral reperfusion and delirium after further adjusting for plasma levels of F2-isoprostanes and isofurans at baseline and ICU admission, the association between hyperoxic cerebral reperfusion and these markers of oxidative injury, and the association between these markers and delirium. RESULTS: Ninety of the 310 patients developed delirium following surgery. Every 10%·hour of intraoperative hyperoxic cerebral reperfusion was independently associated with a 65% increase in the odds of delirium (OR, 1.65 [95% CI, 1.12-2.44]; P=0.01). Hyperoxia prior to ischemia was also independently associated with delirium (1.10 [1.01-1.19]; P=0.02), but hypoxia was not (1.12 [0.97-1.29]; P=0.11). Increased hyperoxic cerebral reperfusion was associated with increased concentrations of F2-isoprostanes and isofurans at ICU admission, increased concentrations of these markers were associated with increased delirium, and the association between hyperoxic cerebral reperfusion and delirium was weaker after adjusting for these markers of oxidative injury. CONCLUSIONS: Intraoperative hyperoxic cerebral reperfusion was associated with increased postoperative delirium, and increased oxidative injury following hyperoxic cerebral reperfusion may partially mediate this association. Further research is needed to assess the potential deleterious role of cerebral hyper-oxygenation during surgery.

Scavengers of reactive γ-ketoaldehydes extend Caenorhabditis elegans lifespan and healthspan through protein-level interactions with SIR-2.1 and ETS-7.

Isoketals (IsoKs) are highly reactive γ-ketoaldehyde products of lipid peroxidation that covalently adduct lysine side chains in proteins, impairing their function. Using C. elegans as a model organism, we sought to test the hypothesis that IsoKs contribute to molecular aging through adduction and inactivation of specific protein targets, and that this process can be abrogated using salicylamine (SA), a selective IsoK scavenger. Treatment with SA extends adult nematode longevity by nearly 56% and prevents multiple deleterious age-related biochemical and functional changes. Testing of a variety of molecular targets for SA's action revealed the sirtuin SIR-2.1 as the leading candidate. When SA was administered to a SIR-2.1 knockout strain, the effects on lifespan and healthspan extension were abolished. The SIR-2.1-dependent effects of SA were not mediated by large changes in gene expression programs or by significant changes in mitochondrial function. However, expression array analysis did show SA-dependent regulation of the transcription factor ets-7 and associated genes. In ets-7 knockout worms, SA's longevity effects were abolished, similar to sir-2.1 knockouts. However, SA dose-dependently increases ets-7 mRNA levels in non-functional SIR-2.1 mutant, suggesting that both are necessary for SA's complete lifespan and healthspan extension.

Controlled moderate hypovolaemia in healthy volunteers is not associated with the development of oxidative stress assessed by plasma F2-isoprostanes and isofurans.

Hypovolaemia can be associated with substantial morbidity, particularly when it occurs in the setting of trauma and in patients with comorbid diseases. Hypovolaemia and inflammation such as occur in the setting of trauma and surgery, are associated with systemic oxidative stress and free-radical injury. Free-radical injury that results from hypovolaemia-induced organ reperfusion may further augment inflammatory processes. It is unknown exactly what proportion of free-radical injury is associated with isolated hypovolaemia as opposed to the contribution from inflammation from surgery or trauma. In the first human study of its kind, we exposed 8 adult male volunteers to venesection-induced hypovolaemia in progressive aliquots of 5% of total blood volume until 20% had been removed. This blood was subsequently reinfused. Plasma F2-isoprostanes and isofurans, markers of in vivo lipid oxidation, were measured by gas chromatography-mass spectrometry at each 5% aliquot venesected and at each 5% reinfused. Between baseline and maximal blood loss there was a minor fall in haemoglobin concentration from 143.9g/l to 138.8g/l (p=0.004, 95% CI 2.2, 8.0g/L). No significant change from baseline occurred in the concentrations of either plasma F2-isoprostanes or isofurans during venesection (p=0.116 and p=0.152, respectively) or blood reinfusion (p=0.553 and p=0.736, respectively). We can conclude that in healthy adult volunteers, isolated hypovolaemia to 20% total blood volume loss is not associated with detectable systemic oxidative stress. The free-radical injury identified in surgical and trauma patients may represent the effects of tissue damage and inflammation, with an uncertain contribution from tissue ischemia as may occur with hypovolaemia.

Mitochondrial dysfunction and oxidative stress in patients with chronic kidney disease.

Mitochondria abnormalities in skeletal muscle may contribute to frailty and sarcopenia, commonly present in patients with chronic kidney disease (CKD). Dysfunctional mitochondria are also a major source of oxidative stress and may contribute to cardiovascular disease in CKD We tested the hypothesis that mitochondrial structure and function worsens with the severity of CKD Mitochondrial volume density, mitochondrial DNA (mtDNA) copy number, BNIP3, and PGC1α protein expression were evaluated in skeletal muscle biopsies obtained from 27 subjects (17 controls and 10 with CKD stage 5 on hemodialysis). We also measured mtDNA copy number in peripheral blood mononuclear cells (PBMCs), plasma isofurans, and plasma F2-isoprostanes in 208 subjects divided into three groups: non-CKD (eGFR>60 mL/min), CKD stage 3-4 (eGFR 60-15 mL/min), and CKD stage 5 (on hemodialysis). Muscle biopsies from patients with CKD stage 5 revealed lower mitochondrial volume density, lower mtDNA copy number, and higher BNIP3 content than controls. mtDNA copy number in PBMCs was decreased with increasing severity of CKD: non-CKD (6.48, 95% CI 4.49-8.46), CKD stage 3-4 (3.30, 95% CI 0.85-5.75, P = 0.048 vs. non-CKD), and CKD stage 5 (1.93, 95% CI 0.27-3.59, P = 0.001 vs. non-CKD). Isofurans were higher in patients with CKD stage 5 (median 59.21 pg/mL, IQR 41.76-95.36) compared to patients with non-CKD (median 49.95 pg/mL, IQR 27.88-83.46, P = 0.001), whereas F2-isoprostanes did not differ among groups. Severity of CKD is associated with mitochondrial dysfunction and markers of oxidative stress. Mitochondrial abnormalities, which are common in skeletal muscle from patients with CKD stage 5, may explain the muscle dysfunction associated with frailty and sarcopenia in CKD Further studies are required to evaluate mitochondrial function in vivo in patients with different CKD stages.

The relationship between plasma lipid peroxidation products and primary graft dysfunction after lung transplantation is modified by donor smoking and reperfusion hyperoxia.

BACKGROUND: Donor smoking history and higher fraction of inspired oxygen (FIO2) at reperfusion are associated with primary graft dysfunction (PGD) after lung transplantation. We hypothesized that oxidative injury biomarkers would be elevated in PGD, with higher levels associated with donor exposure to cigarette smoke and recipient hyperoxia at reperfusion. METHODS: We performed a nested case-control study of 72 lung transplant recipients from the Lung Transplant Outcomes Group cohort. Using mass spectroscopy, F2-isoprostanes and isofurans were measured in plasma collected after transplantation. Cases were defined in 2 ways: grade 3 PGD present at day 2 or day 3 after reperfusion (severe PGD) or any grade 3 PGD (any PGD). RESULTS: There were 31 severe PGD cases with 41 controls and 35 any PGD cases with 37 controls. Plasma F2-isoprostane levels were higher in severe PGD cases compared with controls (28.6 pg/ml vs 19.8 pg/ml, p = 0.03). Plasma F2-isoprostane levels were higher in severe PGD cases compared with controls (29.6 pg/ml vs 19.0 pg/ml, p = 0.03) among patients reperfused with FIO2 >40%. Among recipients of lungs from donors with smoke exposure, plasma F2-isoprostane (38.2 pg/ml vs 22.5 pg/ml, p = 0.046) and isofuran (66.9 pg/ml vs 34.6 pg/ml, p = 0.046) levels were higher in severe PGD compared with control subjects. CONCLUSIONS: Plasma levels of lipid peroxidation products are higher in patients with severe PGD, in recipients of lungs from donors with smoke exposure, and in recipients exposed to higher Fio2 at reperfusion. Oxidative injury is an important mechanism of PGD and may be magnified by donor exposure to cigarette smoke and hyperoxia at reperfusion.

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

BACKGROUND: Cardiopulmonary 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 RESULTS: In 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. CONCLUSIONS: Intravenous acetaminophen attenuates the increase in intraoperative plasma isofuran concentrations that occurs during CPB, while urinary markers were unaffected. TRIAL REGISTRATION: ClinicalTrials.gov NCT01366976.

Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid ß1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

The isoprostanes--25 years later.

Isoprostanes (IsoPs) are prostaglandin-like molecules generated independent of the cyclooxygenase (COX) by the free radical-induced peroxidation of arachidonic acid. The first isoprostane species discovered were isomeric to prostaglandin F2α and were thus termed F2-IsoPs. Since the initial discovery of the F2-IsoPs, IsoPs with differing ring structures have been identified as well as IsoPs from different polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexanenoic acid. The discovery of these molecules in vivo in humans has been a major contribution to the field of lipid oxidation and free radical research over the course of the past 25 years. These molecules have been determined to be both biomarkers and mediators of oxidative stress in numerous disease settings. This review focuses on recent developments in the field with an emphasis on clinical research. Special focus is given to the use of IsoPs as biomarkers in obesity, ischemia-reperfusion injury, the central nervous system, cancer, and genetic disorders. Additionally, attention is paid to diet and lifestyle factors that can affect endogenous levels of IsoPs. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance."

Mice deficient in the gene for cytochrome P450 (CYP)1A1 are more susceptible than wild-type to hyperoxic lung injury: evidence for protective role of CYP1A1 against oxidative stress.

Hyperoxia contributes to acute lung injury in diseases such as acute respiratory distress syndrome in adults and bronchopulmonary dysplasia in premature infants. Cytochrome P450 (CYP)1A1 has been shown to modulate hyperoxic lung injury. The mechanistic role(s) of CYP1A1 in hyperoxic lung injury in vivo is not known. In this investigation, we hypothesized that Cyp1a1(-/-) mice would be more susceptible to hyperoxic lung injury than wild-type (WT) mice, and that the protective role of CYP1A1 is in part due to CYP1A1-mediated decrease in the levels of reactive oxygen species-mediated lipid hydroperoxides, e.g., F2-isoprostanes/isofurans, leading to attenuation of oxidative damage. Eight- to ten-week-old male WT (C57BL/6J) or Cyp1a1(-/-) mice were exposed to hyperoxia (>95% O2) or room air for 24-72 h. The Cyp1a1(-/-) mice were more susceptible to oxygen-mediated lung damage and inflammation than WT mice, as evidenced by increased lung weight/body weight ratio, lung injury, neutrophil infiltration, and augmented expression of IL-6. Hyperoxia for 24-48 h induced CYP1A expression at the mRNA, protein, and enzyme levels in liver and lung of WT mice. Pulmonary F2-isoprostane and isofuran levels were elevated in WT mice after hyperoxia for 24 h. On the other hand, Cyp1a1(-/-) mice showed higher levels after 48-72 h of hyperoxia exposure compared to WT mice. Our results support the hypothesis that CYP1A1 protects against hyperoxic lung injury by decreasing oxidative stress. Future research could lead to the development of novel strategies for prevention and/or treatment of acute lung injury.

Acetaminophen attenuates lipid peroxidation in children undergoing cardiopulmonary bypass.

OBJECTIVE: Hemolysis, occurring during cardiopulmonary bypass, is associated with lipid peroxidation and postoperative acute kidney injury. Acetaminophen inhibits lipid peroxidation catalyzed by hemeproteins and in an animal model attenuated rhabdomyolysis-induced acute kidney injury. This pilot study tests the hypothesis that acetaminophen attenuates lipid peroxidation in children undergoing cardiopulmonary bypass. DESIGN: Single-center prospective randomized double-blinded study. SETTING: University-affiliated pediatric hospital. PATIENTS: Thirty children undergoing elective surgical correction of a congenital heart defect. INTERVENTIONS: Patients were randomized to acetaminophen (OFIRMEV [acetaminophen] injection; Cadence Pharmaceuticals, San Diego, CA) or placebo every 6 hours for four doses starting before the onset of cardiopulmonary bypass. MEASUREMENT AND MAIN RESULTS: Markers of hemolysis, lipid peroxidation (isofurans and F2-isoprostanes), and acute kidney injury were measured throughout the perioperative period. Cardiopulmonary bypass was associated with a significant increase in free hemoglobin (from a prebypass level of 9.8 ± 6.2 mg/dL to a peak of 201.5 ± 42.6 mg/dL postbypass). Plasma and urine isofuran and F2-isoprostane concentrations increased significantly during surgery. The magnitude of increase in plasma isofurans was greater than the magnitude in increase in plasma F2-isoprostanes. Acetaminophen attenuated the increase in plasma isofurans compared with placebo (p = 0.02 for effect of study drug). There was no significant effect of acetaminophen on plasma F2-isoprostanes or urinary makers of lipid peroxidation. Acetaminophen did not affect postoperative creatinine, urinary neutrophil gelatinase-associated lipocalin, or prevalence of acute kidney injury. CONCLUSION: Cardiopulmonary bypass in children is associated with hemolysis and lipid peroxidation. Acetaminophen attenuated the increase in plasma isofuran concentrations. Future studies are needed to establish whether other therapies that attenuate or prevent the effects of free hemoglobin result in more effective inhibition of lipid peroxidation in patients undergoing cardiopulmonary bypass.

Influence of dietary fat type on benzo(a)pyrene [B(a)P] biotransformation in a B(a)P-induced mouse model of colon cancer.

In the US alone, around 60,000 lives/year are lost due to colon cancer. Diet and environment have been implicated in the development of sporadic colon tumors. The objective of this study was to determine how dietary fat potentiates the development of colon tumors through altered B(a)P biotransformation, using the Adenomatous polyposis coli with Multiple intestinal neoplasia mouse model. Benzo(a)pyrene was administered to mice through tricaprylin, and unsaturated (USF; peanut oil) and saturated (SF; coconut oil) fats at doses of 50 and 100 µg/kg via oral gavage over a 60-day period. Blood, colon, and liver were collected at the end of exposure period. The expression of B(a)P biotransformation enzymes [cytochrome P450 (CYP)1A1, CYP1B1 and glutathione-S-transferase] in liver and colon were assayed at the level of protein, mRNA and activities. Plasma and tissue samples were analyzed by reverse phase high-performance liquid chromatography for B(a)P metabolites. Additionally, DNA isolated from colon and liver tissues was analyzed for B(a)P-induced DNA adducts by the (32)P-postlabeling method using a thin-layer chromatography system. Benzo(a)pyrene exposure through dietary fat altered its metabolic fate in a dose-dependent manner, with 100 µg/kg dose group registering an elevated expression of B(a)P biotransformation enzymes, and greater concentration of B(a)P metabolites, compared to the 50 µg/kg dose group (P<.05). This effect was more pronounced for SF group compared to USF group (P<.05). These findings establish that SF causes sustained induction of B(a)P biotransformation enzymes and extensive metabolism of this toxicant. As a consequence, B(a)P metabolites were generated to a greater extent in colon and liver, whose concentrations also registered a dose-dependent increase. These metabolites were found to bind with DNA and form B(a)P-DNA adducts, which may have contributed to colon tumors in a subchronic exposure regimen.

Major metabolite of F2-isoprostane in urine may be a more sensitive biomarker of oxidative stress than isoprostane itself.

BACKGROUND: There is limited literature on the contributors to isoprostane metabolite 2,3-dinor-5,6-dihydro-15-F(2t)-isoprostane (15-F(2t)-IsoP-M) compared with F(2)-isoprostanes (F(2)-IsoPs) as an oxidative stress biomarker. OBJECTIVE: The objective of this study was to investigate whether plasma concentrations of antioxidants, urinary excretion rates of polyphenols, and antioxidants in food and dietary supplements are attributable to both urinary F(2)-IsoP and 15-F(2t)-IsoP-M concentrations. DESIGN: Dietary intake information and blood and urine samples were obtained from 845 healthy middle-aged and elderly female participants of the Shanghai Women's Health Study. Urinary isoprostanes (F(2)-IsoPs and 15-F(2t)-IsoP-M) were measured and adjusted for creatinine concentrations. RESULTS: Urinary 15-F(2t)-IsoP-M and F(2)-IsoP concentrations were lower in subjects who used a multivitamin. Lower F(2)-IsoP concentrations were observed in ginseng users, whereas lower concentrations of 15-F(2t)-IsoP-M were shown in subjects who used a vitamin E supplement. Plasma concentrations of several antioxidants (ie, ß-carotenes, both trans and cis ß-carotenes, lycopene other than trans, 5-cis and 7-cis isomers, cis anhydrolutein, and cis ß-cryptoxanthin) were inversely associated with 15-F(2t)-IsoP-M but not with F(2)-IsoPs, whereas ß-, γ-, and δ-tocopherols were positively associated with 15-F(2t)-IsoP-M but not with F(2)-IsoPs. Urinary polyphenol quercetin was positively associated with both F(2)-IsoPs and 15-F(2t)-IsoP-M. CONCLUSION: The results suggest that the F(2)-IsoP major metabolite 15-F(2t)-IsoP-M may be a more sensitive marker of endogenous oxidative stress status than are F(2)-IsoPs in the assessment of effects of antioxidants on age-related diseases.

Hemoglobin attenuates the effects of inspired oxygen on plasma isofurans in humans during upper-limb surgery.

Reperfusion injury is characterized by significant oxidative stress. F(2)-isoprostanes (F(2)-IsoP's) and isofurans (IsoF's), the latter preferentially produced during increased oxygen tension, are recognized markers of in vivo oxidative stress. We aimed to determine whether increasing oxygen tension during reperfusion modified levels of plasma total IsoF's and F(2)-IsoP's. Forty-five patients undergoing upper-limb surgery were randomized to receive inspired oxygen concentrations of 30, 50, or 80% during the last 15 min of surgery. Venous blood samples were taken before the change in inspired oxygen, after 10 min (before reperfusion), and after 15 min (5 min after reperfusion). IsoF's and F(2)-IsoP's were measured by gas chromatography-mass spectrometry. Venous oxygen tension and hemoglobin concentrations were also measured. Plasma IsoF and F(2)-IsoP levels in the 50 and 80% O(2) groups were not significantly different from those of the 30% O(2) group. In secondary analyses, using data combining all groups, levels of IsoF's, but not F(2)-IsoP's, associated with higher venous oxygen tension (P=0.038). Hemoglobin negatively modified the influence of oxygen tension on levels of IsoF's (P=0.014). This study has shown, for the first time, that plasma IsoF levels associate with higher oxygen tension in a human model of reperfusion, and this effect is significantly attenuated by hemoglobin.

Oxidative stress measured by urine F2-isoprostane level is associated with prostate cancer.

PURPOSE: Oxidative stress is implicated in prostate cancer by several lines of evidence. We studied the relationship between the level of F2-isoprostanes, a validated biomarker of oxidative stress, and prostate cancer and high grade prostatic intraepithelial neoplasia. MATERIALS AND METHODS: This case-control analysis within the Nashville Men's Health Study included men recruited at prostate biopsy. Body morphometrics, health history and urine were collected from more than 2,000 men before biopsy. F2-isoprostanes were measured by gas chromatography/mass spectrometry within an age matched sample of Nashville Men's Health Study participants that included 140 patients with high grade prostatic intraepithelial neoplasia, 160 biopsy negative controls and 200 prostate cancer cases. Multivariable linear and logistic regression was used to determine the associations between F2-isoprostane level, and high grade prostatic intraepithelial neoplasia and prostate cancer. RESULTS: Mean patient age was 66.9 years (SD 7.2) and 10.1% were nonwhite. Adjusted geometric mean F2-isoprostane levels were higher in patients with prostate cancer (1.82, 95% CI 1.66-2.00) or high grade prostatic intraepithelial neoplasia (1.82, 95% CI 1.68-1.96) than in controls (1.63, 95% CI 1.49-1.78, p <0.001), but were similar across Gleason scores (p = 0.511). The adjusted odds of high grade prostatic intraepithelial neoplasia and prostate cancer increased with increasing F2-isoprostane quartile (p-trend = 0.015 and 0.047, respectively) and the highest F2-isoprostane quartile was associated with significantly increased odds of prostate cancer (OR 2.44, 95% CI 1.17-5.09, p = 0.017). CONCLUSIONS: Pre-diagnosis urine F2-isoprostane level is increased in men with high grade prostatic intraepithelial neoplasia or prostate cancer, suggesting urinary F2-isoprostane provides a biomarker for the role for oxidative stress in prostate carcinogenesis. F2-isoprostanes may also serve to estimate the efficacy of interventions targeting oxidative stress mechanisms in prostate cancer prevention or treatment.

Genetic and environmental influences on oxidative damage assessed in elderly Danish twins.

Previous studies have shown an association between oxidative stress and various diseases in humans including cancer, cardiovascular disease, diabetes, and chronic respiratory disease. To what extents this damage is determined by genetic and environmental factors is unknown. In a classical twin study with 198 elderly twins we examined the contributions of genetic versus environmental factors to nucleic acid oxidation and lipid peroxidation. Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and dinor,dihydro F2-isoprostane metabolites (F2-IsoP-M) was measured using liquid chromatography-tandem mass spectrometry. The environmental influence on nucleic acid oxidation and lipid peroxidation was predominant, leaving only little influence from genetic factors, as evidenced by no differences in intraclass correlations between monozygotic (MZ) and dizygotic (DZ) twins, neither for 8-oxodG (r(MZ)=0.55, r(DZ)=0.47; P=0.43), F(2)-IsoP-M (r(MZ)=0.33, r(DZ)=0.22; P=0.42), nor 8-oxoGuo (r(MZ)=0.45, r(DZ)=0.58; P=0.21). Accordingly, heritability estimates for the three markers of oxidative damage were low (h²=0.17-0.22). The three urinary markers of oxidative stress were closely correlated (r=0.60-0.84). In conclusion, we demonstrated in a large population of elderly Danish twins that "whole-body" oxidative damage to nucleic acids and lipids is predominantly determined by potentially modifiable nongenetic factors.

Postoperative acute kidney injury is associated with hemoglobinemia and an enhanced oxidative stress response.

Acute kidney injury (AKI) frequently afflicts patients undergoing cardiopulmonary bypass and independently predicts death. Both hemoglobinemia and myoglobinemia are independent predictors of postoperative AKI. Release of free hemeproteins into the circulation is known to cause oxidative injury to the kidneys. This study tested the hypothesis that postoperative AKI is associated with both enhanced intraoperative hemeprotein release and increased lipid peroxidation assessed by measuring F2-isoprostanes and isofurans. In a case-control study nested within an ongoing randomized trial of perioperative statin treatment and AKI, we compared levels of F2-isoprostanes and isofurans with plasma levels of free hemoglobin and myoglobin in 10 cardiac surgery AKI patients to those of 10 risk-matched controls. Peak plasma free hemoglobin concentrations were significantly higher in AKI subjects (289.0 ± 37.8 versus 104.4 ± 36.5mg/dl, P = 0.01), whereas plasma myoglobin concentrations were similar between groups. The change in plasma F2-isoprostane and isofuran levels (repeated-measures ANOVA, P = 0.02 and P = 0.001, respectively) as well as the change in urine isofuran levels (P = 0.04) was significantly greater in AKI subjects. In addition, change in peak plasma isofuran levels correlated not only with peak free plasma hemoglobin concentrations (r² = 0.39, P = 0.001) but also with peak change in serum creatinine (r² = 0.20, P = 0.01). Postoperative AKI is associated with both enhanced intraoperative hemeprotein release and enhanced lipid peroxidation. The correlations among hemoglobinemia, lipid peroxidation, and AKI indicate a potential role for hemeprotein-induced oxidative damage in the pathogenesis of postoperative AKI.

Tubular epithelial injury and inflammation after ischemia and reperfusion in human kidney transplantation.

OBJECTIVE: To provide an integrated insight into the kinetics of tubular injury, inflammation, and oxidative stress after human kidney transplantation. BACKGROUND: Tissue injury due to ischemia and reperfusion is an inevitable consequence of kidney transplantation. Tubular epithelial injury, inflammation, and oxidative stress play major roles in the pathophysiology of acute kidney injury in small animals, but it remains to be established whether this paradigm holds true for human kidney transplantation. METHODS: Markers of tubular injury, inflammation, and oxidative stress were compared between recipients of kidneys from donors after cardiac death (DCD; N = 8) with prolonged ischemia and recipients of living donor kidneys with minimal ischemia (N = 8). RESULTS: In the early postoperative period, creatinine clearance and tubular sodium reabsorption were profoundly reduced in DCD kidneys, coinciding with significantly increased urinary concentrations of tubular injury markers (neutrophil gelatinase-associated lipocalin, N-acetyl-ß--glucosaminidase, and cystatin C) and an 18-fold increase in renal production of cytokeratin-18, indicating extensive necrotic cell death. Tubular injury in DCD kidneys was followed by greater systemic inflammatory activity and oxidative stress in the postoperative period (measured with 17-plex cytokine arrays and as plasma F2-isoprostanes, respectively). In contrast, no evidence of oxidative damage to either of the 2 kidney types was found in the early reperfusion period. CONCLUSIONS: These findings establish the relevance of observations in animal models for human kidney transplantation and form the basis for development of novel therapies to improve early graft function and expand the use of donor kidneys with prolonged ischemia.

Effects of spinal or general anesthesia on F2-isoprostanes and isofurans during ischemia/reperfusion of the leg in patients undergoing knee replacement surgery.

General and spinal anesthesia are used extensively in orthopedic surgery. However, these methods of anesthesia may result in different amounts of oxygen being delivered to the patient. Ischemia/reperfusion injury after release of the tourniquet initiates free radical-mediated oxidative stress. F2-isoprostanes are reliable markers of in vivo lipid peroxidation. However, under conditions of high oxygen tension, isofurans are formed. We aimed to compare plasma isofurans and F2-isoprostanes in spinal versus general anesthesia in patients undergoing knee-replacement surgery in a randomized, blinded study. Thirty-nine patients were randomized to spinal (SA; n = 19) or general anesthesia (GA; n = 20). Blood was collected before anesthesia, and a tourniquet was then applied to the limb during surgery. After release of the tourniquet, blood samples were collected at 30 min, 2 h, and 24 h for measurement of plasma F2-isoprostanes and isofurans by gas chromatography-mass spectrometry. The two groups were comparable in age and body mass index. Plasma F2-isoprostanes were significantly lower in the GA patients compared with the SA patients (p = 0.045). In contrast, the GA patients had significantly elevated plasma isofurans (p = 0.032). Increased isofurans during GA compared with SA are likely to reflect increased oxidative stress due to elevated oxygen concentrations during GA. Further studies are required to assess the implications of these findings on perioperative outcomes.