A reduction in alcohol consumption is associated with reduced plasma F2-isoprostanes and urinary 20-HETE excretion in men

There is considerable evidence that chronic moderate-to-high alcohol consumption increases blood pressure. The mechanisms by which this occurs are not clear. Alcohol consumption can induce oxidative stress and cytochrome P450 (CYP450) isoforms that are associated with oxidative stress and may influence vascular tone. To study the role of such mechanisms we examined whether reducing alcohol intake in moderate-to-heavy drinkers (40-110 g/day) resulted in changes in urinary excretion of 20-HETE, a CYP450 metabolite of arachidonic acid, and plasma and urinary F(2)-isoprostanes as markers of lipid peroxidation. After a 4-week run-in period during which healthy men maintained their usual drinking pattern they were randomized to a two-way crossover intervention study. In each of the 4-week treatment periods subjects either substituted their usual alcohol intake with a 0.9% alcohol beer or maintained their usual alcohol intake. Plasma and urinary F(2)-isoprostanes and urinary 20-HETE were measured by gas chromatography mass spectrometry, and serum gamma-glutamyl transpeptidase (gamma-GT) was measured as a biomarker of alcohol consumption, at the end of each study period. Sixteen healthy men age 51.0+/-2.7 years and with a BMI of 26.4+/-0.61 kg/m(2) completed the study. The reductions in alcohol intake (72.4+/-5.0 vs 7.9+/-1.6 g/day, p<0.001) and serum gamma-GT (geometric mean 24.4 U/L (95% CI 19.7, 30.2) vs 18.6 U/L (95% CI 15.5, 22.2, p<0.01) were accompanied by a significant fall in blood pressure as well as urinary 20-HETE excretion (158+/-23 vs 109+/-19 pmol/mmol creatinine, p<0.001) and plasma F(2)-isoprostanes (3438+/-158 vs 2929+/-145 pmol/L, p=0.01). A substantial reduction in alcohol consumption in healthy men lowered plasma F(2)-isoprostanes and urinary 20-HETE. Increased oxidative stress and 20-HETE production may be linked, at least in part, to the pathogenesis of alcohol-related hypertension.     

F2-isoprostanes and adiposity in older adults

We examined whether a systemic marker of oxidative stress, F₂‐isoprostanes (F₂‐IPs), was associated with total and regional adiposity, adipocytokines, and change in adiposity. Using data from 726 participants enrolled in the Health, Aging, and Body Composition (Health ABC) study, F₂‐IPs and adipocytokines were measured from baseline plasma samples. Total adiposity was measured by whole‐body dual‐energy X‐ray absorptiometry and regional adiposity by abdominal and thigh computed tomography scans at baseline and 5‐year follow‐up. ANOVA models were estimated to examine associations between F₂‐IP tertiles and baseline adiposity and changes in body composition. Median F₂‐IPs was 54.3 pg/ml; women had significantly higher levels than men (61.5 vs. 48.9 pg/ml, P < 0.001). F₂‐IPs were associated with higher levels of adiponectin, leptin, and tumor necrosis factor‐α (TNF‐α). Positive associations were found between F₂‐IPs and all measures of total and regional adiposity among women. In linear regression models, adipocytokines mediated associations among women. Over 5 years of follow‐up, women in the highest vs. lowest F₂‐IP tertile exhibited significant loss of weight (lowest tertile: ?1.1 kg, highest tertile: ?2.7 kg, P < 0.05). In conclusion, F₂‐IPs were associated with measures of total and regional adiposity in women alone and these associations were partially explained by adipocytokines. F₂‐IPs predicted loss of total adiposity over time among women.     

Elevated F2-isoprostanes in thalassemic patients

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

Effects of aerobic exercise on metabolic syndrome improvement in response to weight reduction

Objective: The objective was to test effects of aerobic exercise training on metabolic syndrome (MetSyn) improvement in response to weight reduction. Research Methods and Procedures: A total of 459 overweight and obese women (age, 49 ± 9 years; BMI, 28 ± 3 kg/m²) were recruited for a baseline examination to test the relationship between cardiorespiratory fitness and metabolic syndrome prevalence; among these, 67 subjects with MetSyn were treated with 14‐week weight‐loss programs, which included low‐calorie diet and aerobic exercise. The MetSyn was defined according to the Examination Committee of Criteria for “Metabolic Syndrome” in Japan. Maximal oxygen uptake (V?o _2max) during a maximal cycling test was measured as an index of cardiorespiratory fitness at baseline and after the intervention. Results: In the baseline examination, age‐ and BMI‐adjusted odds ratios for MetSyn prevalence in the low, middle, and upper thirds of V?o _2max were 1.0 (referent), 0.50 (95% confidence interval, 0.26 to 0.95), and 0.39 (95% confidence interval, 0.14 to 0.96), respectively (linear trend, p = 0.02). The adjusted odds ratios for MetSyn improvement in the two interventions with diet alone and diet plus exercise were 1.0 and 3.68 (95% confidence interval, 1.02 to 17.6; p = 0.04), respectively. Discussion: These results suggest that adding aerobic exercise training to a dietary weight‐reduction program further improves MetSyn (adjusted odds ratio, 3.68) in obese women, compared with diet alone. Further studies on an association between V?o _2max change and MetSyn improvement are needed.     

Oxidative stress and increased formation of vasoconstricting F2-isoprostanes in patients with reversible cerebral vasoconstriction syndrome

The pathophysiology of reversible cerebral vasoconstriction syndrome (RCVS) is unknown. Oxidative stress is detrimental to endothelial function and vascular reactivity. We hypothesized that the oxidative stress marker 8-iso-prostaglandin F_2α, which is also a potent vasoconstrictor, might contribute to the pathogenesis of RCVS. Recruited participants included 103 RCVS patients, 53 patients with primary headache with acute severe attacks, and 54 healthy controls. Subjects recruited prior to 2009 were discovery cohort, whereas those after 2009, replication cohort. Urine samples were obtained from all patients at registration and from 79 patients with RCVS again at remission stage. Urine 8-iso-prostaglandin F_2α was analyzed by liquid chromatography-tandem mass spectrometry. Patients with RCVS received magnetic resonance angiography and transcranial color-coded sonography. In RCVS patients, the urine 8-iso-prostaglandin F_2α level was higher than that in the other groups in discovery, replication, and combined cohorts (RCVS, 0.29±0.18; primary headache with acute severe attacks, 0.21±0.19; control, 0.18±0.09 ng/mg creatinine; P<0.001), and it was positively correlated with the flow velocities of major intracranial arteries, especially within the first week of disease onset (middle cerebral artery, Spearman's correlation coefficient [r_s]=0.580, P=0.002; anterior cerebral artery, r_s=0.472, P=0.042; posterior cerebral artery, r_s=0.457, P=0.022; basilar artery, r_s= 0.530, P=0.002). The 8-iso-prostaglandin F_2α level decreased from the ictalto remission stage in RCVS patients (0.31±0.21 vs 0.16±0.10 ng/mg creatinine, P<0.001). 8-Iso-prostaglandin F_2α was higher in patients with RCVS and correlated with the severity of vasoconstrictions. Further studies are required to explore its potential pathogenic role.     

Postmenopausal hypertension: role of 20-HETE

Blood pressure (BP) increases after menopause. However, the mechanisms responsible have not been elucidated. In this study we tested the hypothesis that 20-hydroxyeicosatetraenoic acids (20-HETE), produced by cytochrome P-450 (CYP450) ω-hydroxylase, contributes to the hypertension in a model of postmenopausal hypertension, aged female spontaneously hypertensive rats (PMR). 1-Aminobenzotriazole, a nonselective inhibitor of arachidonic acid metabolism, for 7 days, reduced BP in PMR but had no effect in young females. Acute intravenous infusion of HET-0016, a specific inhibitor of 20-HETE, over 3 h, also reduced BP in PMR. CYP4A isoform mRNA expression showed no difference in renal CYP4A1 or CYP4A3 but increases in CYP4A2 and decreases in CYP4A8. CYP4A protein expression was decreased in kidney of PMR compared with young females. Endogenous 20-HETE was significantly higher in cerebral vessels of PMR than young females (YF) but was significantly lower in renal vessels of PMR. Omega-hydroxylase activity in cerebral vessels was also higher in PMR but was similar in kidney vessels in both groups. In renal microsomal preparations, endogenous 20-HETE was not different in PMR and young females, but ω-hydroxylase activity was significantly lower in PMR than YF. The data with blockers suggest that 20-HETE contributes to postmenopausal hypertension in SHR. The data also suggest that cerebral production of 20-HETE may be increased and renal tubular production may be decreased in PMR, thus both contributing to their elevated BP.     

F2-isoprostanes are not just markers of oxidative stress

F(2)-isoprostanes are not just markers of oxidative stress. The discovery of F(2)-isoprostanes (F(2)-IsoPs) as specific and reliable markers of oxidative stress in vivo is briefly summarized here. F(2)-IsoPs are also agonists of important biological effects, such as the vasoconstriction of renal glomerular arterioles, the retinal vessel, and the brain microcirculature. In addition to the F(2)-IsoPs, E(2)- and D(2)-IsoPs can be formed by rearrangement of H(2)-IsoP endoperoxides and can give rise to cyclopentenone IsoPs, which are very reactive alpha,beta-unsaturated aldehydes. The same type of reactivity is also shown by acyclic gamma-ketoaldehydes formed as products of the IsoP pathway. Because previous studies suggested a relation between oxidative stress and collagen hyperproduction, it was investigated whether collagen synthesis is induced by F(2)-IsoPs, the most proximal products of lipid peroxidation. In contrast to aldehydes, F(2)-IsoPs act through receptors able to elicit definite signal transduction pathways. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma F(2)-IsoPs were markedly elevated for the entire experimental period; hepatic collagen content was also increased. When hepatic stellate cells from normal liver were cultured up to activation (expression of smooth muscle alpha-actin) and then treated with F(2)-IsoPs in the concentration range found in the in vivo studies (10(-9) to 10(-8) M), a striking increase in DNA synthesis, cell proliferation, and collagen synthesis was observed. Total collagen content was similarly increased. All these stimulatory effects were reversed by the specific antagonist of the thromboxane A(2) receptor, SQ 29 548, whereas the receptor agonist, I-BOP, also had a stimulatory effect. Therefore F(2)-IsoPs generated by lipid peroxidation in hepatocytes may mediate hepatic stellate cell proliferation and collagen hyperproduction seen in hepatic fibrosis.     

F2-isoprostanes as indices of lipid peroxidation in inflammatory diseases

Isoprostanes are a new class of lipids, isomers of conventional enzymatically derived prostaglandins, which are produced in vivo primarily by a free radical-catalyzed peroxidation of polyunsaturated fatty acids. F2-isoprostanes, isomers of the enzyme-derived prostaglandin F2alpha, are the most studied species. Because of their mechanisms of formation, specific structural features that distinguish them from other free radical-generated products and chemical stability, they provide a reliable index of the oxidative component of several diseases in vivo. Consistent data suggest that formation of F2-isoprostanes is indeed altered in a variety of clinical settings associated with inflammation and oxidant stress. Moreover, measurement of F2-isoprostanes might provide a sensitive biochemical basis of dose-selection in studies of natural and synthetic antioxidants.     

Acute angiotensin II increases plasma F2-isoprostanes in salt-replete human hypertensives

Angiotensin (Ang) II induces oxidative stress in vitro and in animal models of hypertension. We tested the hypothesis that Ang II increases oxidative stress in human hypertension, as assessed by plasma F2-isoprostane concentrations. Plasma F2-isoprostanes, hemodynamic and endocrine parameters were measured at baseline and following a 55 min infusion of 3 ng/kg/min Ang II in 13 normotensive and 13 hypertensive volunteers ingesting a high- (200 mmol/d) or low- (10 mmol/d) sodium diet. Mean arterial pressure (MAP) and body mass index were higher in hypertensive subjects. Ang II infusion increased MAP (p<.001) and plasma aldosterone concentrations (p<.001) and decreased plasma renin activity (p<.001) and renal plasma flow (p<.001) to a similar extent in both groups. Plasma F2-isoprostane concentrations were similar at baseline. There was no effect of Ang II on F2-isoprostane concentrations during low-salt intake in either group (normotensive 51.7 +/- 7.1 to 53.7 +/- 6.5 pg/ml and hypertensive 52.2 +/- 8.2 to 56.2 +/- 10.0 pg/ml; mean +/- SE). During high-salt intake, Ang II increased F2-isoprostane concentrations in the hypertensive group (52.3 +/- 7.2 to 63.2 +/- 10.4 pg/ml, p=0.010) but not in the normotensive group (54.2 +/- 4.4 to 58.9 +/- 6.6 pg/ml, p=0.83). Acute Ang II infusion increases oxidative stress in vivo in hypertensive humans. The renin-angiotensin system may contribute to oxidative stress in human cardiovascular disease.     

Increased levels of F2-isoprostanes following aneurysmal subarachnoid hemorrhage in humans

Subarachnoid hemorrhage (SAH) resulting from aneurysmal rupture is the major cause of nontraumatic SAH. We hypothesized that oxidative stress could be increased following aneurysmal SAH due to hemoglobin release and ischemia-reperfusion injury and that may further contribute to poor outcome. We collected plasma and cerebrospinal fluid (CSF) samples from 11 non-SAH controls and 15 aneurysmal SAH patients for up to 10 days after surgery and investigated status of oxidative stress in patients. Results showed that mean or peak levels of F(2)-isoprostanes (F(2)-IsoPs), a specific marker of lipid peroxidation, and total nitrate/nitrite, metabolites of nitric oxide and peroxynitrite, in CSF and plasma were significantly higher in SAH patients than in controls. First-day levels were also higher in CSF, but not in plasma, in SAH patients. Moreover, mean and peak levels of CSF F(2)-IsoPs were positively correlated with poor outcome or severity of clinical conditions in patients. Furthermore, levels of retinol, delta-tocopherol, beta+gamma-tocopherol, lutein, beta-carotene, and coenzyme Q(10) in plasma were significantly lower in SAH patients than in controls. Our results indicate that oxidative damage may play important roles in the severity and complications of aneurysmal SAH and suggest that means to suppress lipid peroxidation may be beneficial in improving the outcome of aneurysmal SAH.     

Quantification of F2-isoprostanes as a biomarker of oxidative stress

Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2alpha)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F2-isoprostanes (F2-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent independent study sponsored by the National Institutes of Health (NIH), F2-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well known biomarkers. This protocol details our laboratory's method to quantify F2-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry (GC-MS). This procedure can be completed for 12-15 samples in 6-8 h.     

The role of 20-HETE in androgen-mediated hypertension

Androgen plays an important role in blood pressure regulation. Epidemiological studies have shown that men have a higher prevalence for developing hypertension than aged-matched, premenopausal women. Interestingly, postmenopausal women and women with polycystic ovary syndrome, both of which have increased endogenous androgen production, have elevated risks for hypertension suggesting that androgen may contribute to its development. Studies from our laboratory and others have provided substantial evidence that 20-hydroxyeicosatetraenoic acid (20-HETE) mediates the hypertension seen in rodents treated with androgen. 20-HETE is the cytochrome P450 (CYP)-derived ω-hydroxylated metabolite of arachidonic acid. 20-HETE plays a complex role in blood pressure regulation. In the kidney tubules, 20-HETE decreases blood pressure by promoting natriuresis, while in the microvasculature it has a pressor effect. In the microcirculation, 20-HETE participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor stimuli and contributes to myogenic, mitogenic and angiogenic responses. In addition, 20-HETE acts on the endothelium to promote endothelial dysfunction and endothelial activation. Recently, we have demonstrated that 20-HETE induces endothelial ACE thus setting forth a potential feed forward mechanism through activation of the renin-angiotensin-aldosterone system. In this review, we will discuss the pro-hypertensive effects of 20-HETE and its role in androgen-induced vascular dysfunction and hypertension.     

Plasma F2-isoprostanes are elevated in newborns and inversely correlated to gestational age

F(2)-isoprostanes, prostaglandin F(2)-like compounds formed by free radical-catalyzed lipid peroxidation, are considered the most reliable markers of oxidative stress. It has been repeatedly suggested that newborns are exposed to conditions of oxidative stress resulting from the change from a low oxygen pressure in utero to a high oxygen pressure at birth. We measured the levels of F(2)-isoprostanes in plasma of newborns by gas chromatography/mass spectrometry and we found that F(2)-isoprostanes are significantly higher in term newborns compared to healthy adults. The greatest values were found in preterm newborns in whom F(2)-isoprostanes are even higher than in term babies. Moreover a significant inverse correlation was found between the plasma levels of isoprostanes and the gestational age. A quite normal level of isoprostanes was found in the mothers both at delivery and during pregnancy. Placental total F(2)-isoprostanes (sum of free plus esterified) were significantly higher in preterm compared to term deliveries and such a difference might account for the difference in plasma isoprostanes. Plasma non-protein-bound iron is higher in preterm than in term newborns, even if no correlation was found with plasma F(2)-isoprostanes. Erythrocyte desferrioxamine-chelatable iron content (0 time) and release (24 h of aerobic incubation) are higher in newborns than in adults and in preterm than in term newborns, but again no correlation was found with plasma F(2)-isoprostanes. The marked increase in plasma isoprostanes suggests that oxidative stress is a feature of the physiopathological changes seen in the perinatal period.     

Lower levels of F2-isoprostanes in serum and livers of long-lived Ames dwarf mice

F₂-isoprostanes (IsoPs), lipid peroxidation products, are markers that quantitatively measure levels of oxidative stress. IsoP levels increase in tissues and serum of aging animals suggesting an increase in oxidative stress. This supports the Free Radical Theory of Aging, which proposes that elevated levels of reactive oxygen species (ROS) cause macromolecular damage, and is a factor in the age-associated decline in tissue function. Numerous studies have shown that the longevity of long-lived mutant mice correlates with their resistance to oxidative stress. However, although the Ames dwarf (DW) mice show resistance to oxidative stress, it has not been shown that these mice have inherently lower levels of ROS. Our results show that the serum and liver IsoP levels in DW mice are lower at all ages suggesting that the lower levels of endogenous ROS production in DW mice may be a factor in their resistance to oxidative stress and longevity.     

F2-isoprostanes as an indicator and risk factor for coronary heart disease

Coronary heart disease (CHD) is the leading single cause of death in the United States and most Western countries, killing more than 400,000 Americans per year. Although CHD often manifests suddenly as a fatal myocardial infarction, the atherosclerosis that gives rise to the infarction develops gradually and can be markedly slowed or even reversed through pharmacological and lifestyle interventions. These same atherosclerotic processes also drive related vascular diseases such as stroke and peripheral artery disease, and individuals surviving occlusive events often develop additional complications including ischemic cardiomyopathy and heart failure. Therefore, better detection of subclinical atherosclerosis, along with more effective treatments, could significantly reduce the rate of death from CHD and related vascular diseases in the United States. In recent years, oxidation of polyunsaturated fatty acids (PUFAs) in plasma lipoproteins has been postulated to be a critical step in the development of atherosclerosis. If so, then monitoring lipid peroxidation should be a useful indicator of disease risk and progression. This review focuses on the evidence that specific PUFA peroxidation products, the F(2)-isoprostanes, are useful biomarkers that could potentially be utilized as indicators of CHD.     

Radioimmunological measurement of F(2)-isoprostanes after hydrolysis of lipids in tissues

8-Iso-prostaglandin F(2 alpha)(8-iso-PGF(2 alpha)) is a major isoprostane formed in vivo mainly by non-enzymatic peroxidation of arachidonic acid and a potential biomarker of oxidative injury. We have recently reported development of a specific radioimmunoassay for the measurement of free 8-iso-PGF(2 alpha)in plasma and urine. The aim of this study was to employ this radioimmunoassay to analyze the total amount of 8-iso-PGF(2 alpha)(sum of free and esterified) in liver tissues by using alkaline hydrolysis, and to apply it in an experimental model of carbon tetrachloride-induced lipid peroxidation in rats. Basal levels of total 8-iso-PGF(2 alpha)in hydrolyzed liver tissues of control rats were 6.5 times higher than the levels of free 8-iso-PGF(2 alpha). At maximum formation of total 8-iso-PGF(2 alpha)in the livers of carbon tetrachloride-treated rats, total levels of 8-iso-PGF(2 alpha)were almost 13 times higher than the levels of free 8-iso-PGF(2 alpha). In conclusion, high levels of 8-iso-PGF(2 alpha)in tissues can be quantified after hydrolysis both at basal conditions and in a model of increased lipid peroxidation. The methodology for measurement of total levels of 8-iso-PGF(2 alpha)in tissues may be suitable for future investigations of the location of oxidative injury in the body.     

Serum selenium predicts levels of F2-isoprostanes and prostaglandin F2alpha in a 27 year follow-up study of Swedish men

Low concentrations of selenium (Se) predict mortality and cardiovascular diseases in some populations. The effect of Se on in vivo indicators of oxidative stress and inflammation, two important features of atherosclerosis, in human populations is largely unexplored. This study investigated the longitudinal association between serum selenium (s-Se) and a golden standard indicator of oxidative stress in vivo (8-iso-prostaglandin F2alpha, a major F2-isoprostane), an indicator of cyclooxygenase (COX)-mediated inflammation (prostaglandin F2alpha), high sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6) and serum amyloid A protein (SAA) in a follow-up study of 27 years. The s-Se was measured in 615 Swedish men at 50 years of age in a health investigation. The status of oxidative stress and inflammation was evaluated in a re-investigation 27 years later by quantification of urinary 8-iso-PGF2alpha and 15-keto-dihydro-PGF2alpha (a major metabolite of PGF2alpha) and serum hsCRP, SAA and IL-6. Men in the highest quartile of s-Se at age 50 had decreased levels of 8-iso-PGF2alpha compared to all lower quartiles and decreased levels of PGF2alpha compared to all lower quartiles at follow-up. These associations were independent of BMI, diabetes, hyperlipidemia, hypertension, smoking, alpha-tocopherol and beta-carotene at baseline. The s-Se was not associated with hsCRP, SAA or IL-6 at follow-up. In conclusion, high concentrations of s-Se predict reduced levels of oxidative stress and subclinical COX-mediated (but not cytokine-mediated) inflammation in a male population. The associations between Se, oxidative stress and inflammation, respectively, might be related to the proposed cardiovascular protective property of Se.     

Measurement of F(2)-isoprostanes unveils profound oxidative stress in aged rats

Free radicals have been theorized to play a causative role in the normal aging process. To date, methods used to detect oxidative stress in aged experimental animals have only detected 2- to 3-fold differences or less between young and aged animals. Measurement of F(2)-isoprostanes has emerged as probably the most reliable approach to assess oxidative stress status in vivo. Therefore, we measured levels of F(2)-isoprostanes free in plasma and levels esterified in plasma lipids in young rats (3-4 months of age) and aged rats (22-24 months of age). Plasma concentrations of free F(2)-isoprostanes were increased dramatically by a mean of 20.3-fold (range 4.3 to 42.9-fold) and levels esterified in plasma lipids were also strikingly increased by a mean of 29.9-fold (range 15.8 to 50.0-fold). These findings unveil profound oxidative stress in aged rats which adds considerable support for the free radical theory of aging.