Changes in urinary dinor dihydro F2-isoprostane metabolite concentrations,a marker of oxidative stress,during and following asthma exacerbations

To investigate changes in oxidant stress during and following acute asthma exacerbations, this stidy measured 2,3-dinor-5,6-dihydro-15-F_2t-IsoP (F₂-IsoP-M), the major urinary metabolite of 15-F_2t-IsoP, in eight asthmatic adults, during and following an asthma hospitalization. F₂-IsoP-M concentrations at admission and follow-up were significantly higher than discharge (admission median: 4.12 ng/Cr mg, range 1.89–7.8; follow-up: 2.47 ng/Cr mg (1.56–6.86); discharge: 1.42 ng/Cr mg (0.7–4.44); both p<0.01), but not significantly different between admission and follow-up. F₂-IsoP-M concentrations at follow-up were higher than a control group with stable asthma (0.68 ng/Cr mg (0.31–1.5), p=0.0008). In conclusion, asthma exacerbations requiring hospitalization are associated with 6-fold higher urinary F₂-IsoP-M concentrations compared to stable asthmatics. F₂-IsoP-M concentrations decreased significantly during hospitalization, but significant elevations 3 months following hospitalization suggest ongoing oxidative stress despite clinical improvement. Urinary F₂-IsoP-M may be a clinically useful, simple non-invasive systemic measure of oxidative stress in asthmatics, providing information not captured by spirometry or symptoms.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

The effect of valproic acid on hepatic and plasma levels of 15-F2t-isoprostane in rats

The mechanism by which valproic acid (VPA) induces liver injury remains unknown, but it is hypothesized to involve the generation of toxic metabolites and/or reactive oxygen species. This study's objectives were to determine the effect of VPA on plasma and hepatic levels of the F(2)-isoprostane, 15-F(2t)-IsoP, a marker for oxidative stress, and to investigate the influence of cytochrome P450- (P450-) mediated VPA biotransformation on 15-F(2t)-IsoP levels in rats. In rats treated with VPA (500 mg/kg), plasma 15-F(2t)-IsoP was increased 2.5-fold at t(max) = 0.5 h. Phenobarbital pretreatment (80 mg/kg/d for 4 d) in VPA-treated rats increased plasma and liver levels of free 15-F(2t)-IsoP by 5-fold and 3-fold, respectively, when compared to control groups. This was accompanied by an elevation in plasma and liver levels of P450-mediated VPA metabolites. Pretreatment with SKF-525A (80 mg/kg) or 1-aminobenzotriazole (100 mg/kg), which inhibited P450-mediated VPA metabolism, did not attenuate the increased levels of plasma 15-F(2t)-IsoP in VPA-treated groups. Plasma and hepatic levels of 15-F(2t)-IsoP were further elevated after 14 d of VPA treatment compared to single-dose treatment. Our data indicate that VPA increases plasma and hepatic levels of 15-F(2t)-IsoP and this effect can be enhanced by phenobarbital by a mechanism not involving P450-catalyzed VPA biotransformation.     

Correlation between two markers of inflammation, serum C-reactive protein and interleukin 6, and indices of oxidative stress in patients with high risk of cardiovascular disease.

As evidence of the involvement of inflammation and oxidative damage in pathogenesis of age-related chronic diseases is growing, epidemiologists need to develop measures of both conditions to study their relationships in human populations. One way of searching for appropriate biomarkers is to examine correlations between different inflammatory markers and oxidative indices. We examined cross-sectional correlations between two inflammatory markers, serum C-reactive protein (CRP) and interleukin (IL)-6, and three oxidative indices, plasma levels of alpha-tocopherol and beta-carotene, and urinary levels of 2,3-dinor-5,6-dihydro-15-F2t-isoprostane (F2-IsoP), in 60 individuals at high risk of cardiovascular disease. Correlations between the biomarkers were examined graphically and using the Pearson correlation coefficient. No correlation was found between plasma levels of alpha-tocopherol and either of the inflammatory markers. Plasma beta-carotene inversely correlated with IL-6 (r = -0.46, p=0.0002) and CRP (r = -0.41, p = 0.001). Although urinary F2-IsoP did not correlate with IL-6, this biomarker positively correlated with CRP (r = 0.31, p = 0.002). As only urinary F2-IsoP levels have been validated against known oxidative assaults, their positive association with CRP levels is interpreted as evidence of an interconnection between low-level inflammation and oxidative status. Urinary levels of F2-IsoP and serum levels of CRP represent appropriate biomarkers for future studies of inflammation and oxidative status in humans.     

Oxidant stress but not thromboxane decreases with epoprostenol therapy

Epoprostenol has improved the outcome of patients with primary pulmonary hypertension (PPH); however, its mechanism of action remains poorly understood. Isoprostanes are easily measured markers of oxidant stress and can activate platelets leading to increased thromboxane A2 (TxA2) production. We hypothesized that oxidant stress is associated with increased TxA2 synthesis and that epoprostenol decreases oxidant stress and TxA2 production in patients with PPH. Morning urine samples were obtained from 19 patients with PPH. We measured urinary metabolites of the isoprostane, 8-iso-PGF2alpha (F2-IsoP-M), and of TxA2 (Tx-M) before and after treatment with epoprostenol in patients with PPH. Mean (+/-SE) levels of F2-IsoP-M were elevated at baseline in our patients, 863 +/- 97 pg/mg creatinine. During treatment with epoprostenol, values decreased to 636 +/- 77 pg/mg creatinine (P = 0.011), and there was a strong correlation between the change in F2-IsoP-M and follow-up pulmonary vascular resistance (R2 = 0.69, P < 0.001). Tx-M levels were markedly elevated at baseline and were unchanged with therapy. These results indicate that oxidant stress decreases with epoprostenol therapy and is associated with hemodynamic and clinical improvement. The failure of Tx-M to decrease with therapy suggests that epoprostenol does not exert a beneficial effect through inhibition of TxA2 production in patients with PPH.     

Induction of biotransformation enzymes by the carcinogenic air-pollutant 3-nitrobenzanthrone in liver, kidney and lung, after intra-tracheal instillation in rats

We investigated the effect of the seasonal variability of environmental air pollutants on oxidative stress and cytogenetic biomarkers in a group of 59 city policemen working in Prague, Czech Republic. The studied group was monitored in February and May 2007. The exposure to environmental pollutants (carcinogenic polycyclic aromatic hydrocarbons, c-PAHs, including benzo[a]pyrene, B[a]P, and particulate matter of aerodynamic diameter<2.5μm, PM2.5) was measured by personal and/or stationary monitors. Levels of c-PAHs were significantly higher in winter than spring, while exposure to PM2.5 was higher in May than in February 2007. We did not observe any significant difference between the two seasons for any biomarker of oxidative stress (8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG, 15-F(2t)-isoprostane, 15-F(2t)-IsoP, protein carbonyl levels) or any cytogenetic parameter, including the genomic frequency of translocations (F(G)/100), the percentage of aberrant cells (%AB.C.) or the number of acentric fragments (ace). Analyses of associations between oxidative stress biomarkers and cytogenetic parameters showed a negative relationship between protein oxidation and F(G)/100, as well as protein oxidation and ace. We further analyzed the effect of air pollution on all subjects regardless of the season. Data from stationary monitors showed that 8-oxodG levels were significantly increased by exposure to PM2.5 over a 2-day period before sampling and by exposure to B[a]P over a 28-day period, days 57-84 before sampling. 15-F(2t)-IsoP levels were increased after exposure to B[a]P over both 2-day and 3-day periods preceding sample collection and after exposure to c-PAHs over a 2-day period before sampling. %AB.C. was significantly affected by exposure to B[a]P over a 14-day period, days 57-70 before sampling. In summary, our results indicate that the exposure to environmental pollutants affects urinary excretion of 8-oxodG, lipid peroxidation and the frequency of chromosomal aberrations.     

The effects of oxidation products of arachidonic acid and n3 fatty acids on vascular and platelet function

15-F?(t)-isoprostane (15-F?(t)-IsoP), an oxidation product of arachidonic acid (AA), affects vascular and platelet function; however, the bioactivity of other fatty acids oxidation products is unknown. This paper studied rat aortic vascular reactivity and human platelet aggregation in response to 14 oxidation products of AA, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and α-linolenic acid (ALA) compared with 15-F?(t)-IsoP. It also compared the F?(t)-IsoPs profile in human platelets. EPA-derived 15-F?(t)-IsoP constricted rat aorta less than 15-F?(t)-IsoP, but none of the other oxidation products affected vascular reactivity. Only 15-F?(t)-IsoP (10?? M) directly affected platelet aggregation. 15-F?(t)-IsoP, ent-16-F?-phytoprostane (from ALA) and isofurans A and B (from AA) inhibited reversible aggregation to U46619. Unlike plasma, the platelet profile of F?-IsoP showed that 8-F(2t)-IsoP were higher than 15-F?(t)-IsoP. Unlike 15-F?(t)-IsoP, the test compounds derived from fatty acids oxidation did not affect vascular or platelet function. Elevated platelet 8-F?(t)-IsoP could limit 15-F?(t)-IsoP-induced aggregation under conditions of oxidant stress.     

Oxidative and nitrosative stress markers in bus drivers

Exposure to ambient air pollution is associated with many diseases. Oxidative and nitrosative stress are believed to be two of the major sources of particulate matter (PM)-mediated adverse health effects. PM in ambient air arises from industry, local heating, and vehicle emissions and poses a serious problem mainly in large cities. In the present study we analyzed the level of oxidative and nitrosative stress among 50 bus drivers from Prague, Czech Republic, and 50 matching controls. We assessed simultaneously the levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP) and 8-oxodeoxyguanosine (8-oxodG) in urine and protein carbonyl groups and 3-nitrotyrosine (NT) in blood plasma. For the analysis of all four markers we used ELISA techniques. We observed significantly increased levels of oxidative and nitrosative stress markers in bus drivers. The median levels (min, max) of individual markers in bus drivers versus controls were as follows: 8-oxodG: 7.79 (2.64-12.34)nmol/mmol versus 6.12 (0.70-11.38)nmol/mmol creatinine (p<0.01); 15-F(2t)-IsoP: 0.81 (0.38-1.55)nmol/mmol versus 0.68 (0.39-1.79)nmol/mmol creatinine (p<0.01); carbonyl levels: 14.1 (11.8-19.0)nmol/ml versus 12.9 (9.8-16.6)nmol/ml plasma (p<0.001); NT: 694 (471-3228)nmol/l versus 537 (268-13833)nmol/l plasma (p<0.001). 15-F(2t)-IsoP levels correlated with vitamin E (R=0.23, p<0.05), vitamin C (R=-0.33, p<0.01) and cotinine (R=0.47, p<0.001) levels. Vitamin E levels also positively correlated with 8-oxodG (R=0.27, p=0.01) and protein carbonyl levels (R=0.32, p<0.001). Both oxidative and nitrosative stress markers positively correlated with PM2.5 and PM10 exposure. In conclusion, our study indicates that exposure to PM2.5 and PM10 results in increased oxidative and nitrosative stress.     

Urinary F₂-isoprostanes, obesity, and weight gain in the IRAS cohort

Obesity has been associated with increased F(2)-isoprostane (F(2)-IsoP) levels cross-sectionally. However, the prospective association may be inverse, based on our earlier finding that elevated urinary F(2)-IsoP levels predict lower risk of diabetes. This earlier finding led us to hypothesize that urinary F(2)-IsoPs reflect the intensity of oxidative metabolism and as such predict lower risk of both diabetes and weight gain. We examined cross-sectional relationships with obesity and prospective relationships with weight gain using the data from 299 participants of the Insulin Resistance Atherosclerosis Study (IRAS), all of whom were free of diabetes at baseline. Four urinary F(2)-IsoPs were assayed in stored baseline urine samples using liquid chromatography with tandem mass spectrometry: iPF(2α)-III, 2,3-dinor-iPF(2α)-III, iPF(2α)-VI, and 8,12-iso-iPF(2α)-VI (F(2)-IsoP 1-4, respectively). Baseline F(2)-IsoPs were positively associated with baseline measures of obesity; the strongest associations were found with two F(2)-IsoPs: odds ratios (95% confidence intervals) for overall and abdominal obesity were 1.74 (1.26-2.40) and 1.63 (1.18-2.24) for F(2)-IsoP2 and 1.47 (1.12-1.94) and 1.64 (1.22-2.20) for F(2)-IsoP4. F(2)-IsoP2 showed the strongest and significant inverse association with weight gain during the 5-year follow-up period: increase in F(2)-IsoP2 equal to 1 s.d. was associated with 0.90 kg lower weight gain (P = 0.02) and the odds ratios for relative (≥5%) and absolute (≥5 kg) weight gain were 0.67 (0.47-0.96) and 0.57 (0.37-0.87), respectively. The other three F(2)-IsoPs were consistently inversely associated with weight gain, although not significantly, suggesting that different F(2)-IsoPs vary in their ability to detect the association with weight gain.     

Effects of short-term treatment with vitamin E in systemic sclerosis: a double blind, randomized, controlled clinical trial of efficacy based on urinary isoprostane measurement

This double blind randomized controlled trial was designed to investigate whether short-term vitamin E treatment at doses of 500 and 1000 mg/day, compared to placebo, decreased urinary F(2)-isoprostanes and improved the microvascular perfusion after cold exposure in patients suffering from SSc. Thirty-three eligible patients were randomly assigned in a 1.3:1:1 ratio to receive placebo, vitamin E 500 mg, or vitamin E 1000 mg daily for 3 weeks. Clinical examination, analysis of plasma vitamin E, urinary F(2)-isoprostane levels and a whole body cooling test were performed at baseline and after a 3-week period of treatment. Urinary 15-F(2t)-IsoP levels and cutaneous blood flow variation in response to cold did not significantly differ before versus after treatment in any group. Furthermore, no difference was found between groups after 3 weeks of treatment. We show that 3-week vitamin E treatment at doses of 500 or 1000 mg/day neither decreases the basal rate of lipid peroxidation nor improves microvascular perfusion after cold exposure. These data does not support the need for phase III clinical trials to test efficacy of vitamin E in SSc.     

15-F(2t)-isoprostane exacerbates myocardial ischemia-reperfusion injury of isolated rat hearts

Reactive oxygen species induce formation of 15-F(2t)-isoprostane (15-F(2t)-IsoP), a specific marker of in vivo lipid peroxidation, which is increased after myocardial ischemia and during the subsequent reperfusion. 15-F(2t)-IsoP possesses potent bioactivity under pathophysiological conditions. However, it remains unknown whether 15-F(2t)-IsoP, by itself, can influence myocardial ischemia-reperfusion injury (IRI). Adult rat hearts were perfused by the Langendorff technique with Krebs-Henseleit (KH) solution at a constant flow rate of 10 ml/min. 15-F(2t)-IsoP (100 nM), SQ-29548 (1 microM, SQ), a thromboxane receptor antagonist that can abolish the vasoconstrictor effect of 15-F(2t)-IsoP, 15-F(2t)-IsoP + SQ in KH, or KH alone (vehicle control) was applied for 10 min before induction of 40 min of global ischemia followed by 60 min of reperfusion. During ischemia, saline (control), 15-F(2t)-IsoP, 15-F(2t)-IsoP + SQ, or SQ in saline was perfused through the aorta at 60 microl/min. 15-F(2t)-IsoP, 15-F(2t)-IsoP + SQ, or SQ in KH was infused during the first 15 min of reperfusion. Coronary effluent endothelin-1 concentrations were significantly higher in the group treated with 15-F(2t)-IsoP than in the control group during ischemia and also in the later phase of reperfusion (P < 0.05). Infusion of 15-F(2t)-IsoP increased release of cardiac-specific creatine kinase, reduced cardiac contractility during reperfusion, and increased myocardial infarct size relative to the control group. SQ abolished the deleterious effects of 15-F(2t)-IsoP. 15-F(2t)-IsoP exacerbates myocardial IRI and may, therefore, act as a mediator of IRI. 15-F(2t)-IsoP-induced endothelin-1 production during cardiac reperfusion may represent a mechanism underlying the deleterious actions of 15-F(2t)-IsoP.     

Chromium intake and urinary chromium excretion of trauma patients

Glucose metabolism is altered after trauma and those factors that affect glucose metabolism often affect chromium (Cr) metabolism and excretion. To ascertain whether urinary Cr excretion is affected by the elevated serum glucose and other factors associated with trauma, the serum glucose and urinary Cr and Creatinine (Cre) excretion of seven severely traumatized patients were determined. The Cr concentration of intravenous (IV) fluids administered was determined and approximate Cr intake calculated. For all patients, urinary Cr concentration was high in the initial sample collected within 24 h of admission (10.3 ± 2.5 ng/mL, mean ± SEM) and decreased significantly (P < 0.05) by 42 h (2.0 ±0.6 ng/mL). The mean urinary Cr concentration 42 h following admission was 10 times greater than the urinary Cr concentration of normal, healthy subjects (0.2 ± 0.02 ng/mL). There was no significant change in urinary Cre concentration within 42 h of admission, therefore the ratio of urinary Cr to Cre (ng Cr:mg Cre) also decreased. Serum glucose concentration was elevated at admission (170 ± 18 mg/dL, mean ± SD) and decreased to 145 ± 10 mg/dL by 48 h post-admission. The intravenous fluids, dextrose and NaCl, were the lowest in Cr of the samples tested, range 0.02 to 0.20 ng/mL; lactated Ringer’s solution, with or without dextrose, contained 10-20 times more Cr and plasma protein fraction contained approximately 32 ng/mL. The mean calculated Cr intake for the first 24 h postadmission was 37.1 µg/d, significantly greater (P < 0.01) than intake from 24 to 48 h (0.12 µg/d) and 48-72 h (1.63 µg/d). The IV intake of Cr varied for trauma patients depending on fluids required during treatment, but for all patients the relatively high IV Cr intake was rapidly excreted in the urine. These data demonstrate that urinary Cr concentration is elevated several-fold within 24 h of trauma and that Cr contents of intravenous fluids administered in the days immediately following injury vary dramatically. The effects of trauma alone on Cr excretion are difficult to assess because of the variable intake of Cr from IV fluids.     

Isomer-specific contractile effects of a series of synthetic f2-isoprostanes on retinal and cerebral microvasculature

F2-isoprostanes (F2-IsoP's) are biologically active prostanoids formed by free radical-mediated peroxidation of arachidonic acid. Four different F2-IsoP regioisomers (5-, 8-, 12-, and 15-series), each comprising eight racemic diastereomers, total 64 compounds. Information regarding the biological activity of IsoP's is largely limited to 15-F2t-IsoP (8-iso-PGF2alpha). We recently demonstrated that 15-F2t-IsoP and its metabolite, 2,3-dinor-5,6-dihydro-15-F2t-IsoP, evoked vasoconstriction and TXA2 generation in retina and brain microvasculature. We have now examined and compared the biological activities of a series of recently synthesized new 5-, 12-, and 15-series F2-IsoP isomers in pig retinal and brain microvasculature. We hereby show that other 15-series F2-IsoP isomers, 15-epi-15-F2t-IsoP, ent-15-F2t-IsoP, and ent-15-epi-15-F2t-IsoP, are also potent vasoconstrictors. The 12-series isomers tested, 12-F2t-IsoP and 12-epi-12-F2t-IsoP, also caused marked vasoconstriction. Of the 5-series isomers tested, 5-F2t-IsoP and 5-epi-5-F2t-IsoP possessed no vasomotor properties, whereas ent-5-F2t-IsoP caused modest vasoconstriction. The vasoconstriction of ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP was abolished by removal of the endothelium, by TXA2 synthase and receptor inhibitor (CGS12970, L670,596), and by receptor-mediated Ca2+ channel blockade (SK & F96365); correspondingly, these isomers increased TXB2 formation by activating Ca2+ influx (detected with fura 2-AM) through non-voltage-dependent receptor-mediated Ca2+ entry (SK & F96365 sensitive) in endothelial cells. In conclusion, as seen with 15-F2t-IsoP, ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP constricted both retinal and brain microvessels by inducing endothelium-dependent TXA2 synthesis. These new findings broaden the scope of our understanding regarding the potential involvement of F2-IsoP's as mediators of oxidant injury.     

Lack of effect of acute oral ingestion of vitamin C on oxidative stress, arterial stiffness or blood pressure in healthy subjects

Vitamin C is a potent antioxidant in vitro and has been reported to act as a vasodilator, possibly by increasing nitric oxide bioavailability. This study examined the antioxidant and vascular effects of a single large oral dose of vitamin C in 26 healthy human volunteers. Haemodynamic and oxidative DNA and lipid damage markers were measured for 8 h following an oral dose of 2 g vitamin C or placebo. Vitamin C had no effect on vasodilation (measured by augmentation index (mean change=0.04%, 90% CI=- 2.20% to 2.28%) or forearm blood flow (-0.19%/min (-0.68, 0.30)), in comparison to placebo) or on several markers of oxidative stress including DNA base oxidation products in blood cells, 8-hydroxy-2'-deoxyguanosine (8O HdG) in urine (0.068 (-0.009, 0.144)) or urinary or plasma total F(2)-isoprostanes (-0.005 ng/ml (-0.021, 0.010), -0.153 ng/mg (-0.319, 0.014), respectively).     

Postocclusive reactive hyperemia inversely correlates with urinary 15-F2t-isoprostane levels in systemic sclerosis

Microvascular dysfunction and increased oxidative stress are major hallmarks of the systemic sclerosis disease process. The primary objective of this study was to test whether there is a link between peak postocclusive hyperemia and urinary levels of the F2-isoprostane 15-F2t-IsoP (8-iso-PGF2alpha) in patients suffering from systemic sclerosis. We enrolled 43 patients suffering from systemic sclerosis, 33 patients with primary Raynaud's phenomenon (RP), and 25 healthy volunteers. Microvascular function was assessed using the postocclusive hyperemia monitored by laser Doppler flowmetry. Endothelium-independent response was monitored after 0.4 mg sublingual nitroglycerin. Oxidative stress status was assessed by urinary levels of the F2-isoprostane 15-F2t-IsoP using GC-MS. The peak postocclusive vascular conductance was altered in subjects with systemic sclerosis and primary RP compared to controls (respectively 28 (7-48), 30 (13-48), and 39.9 (13-63) mV/mm Hg, p = 0.01). F2-isoprostanes were increased in the systemic sclerosis group compared to primary Raynaud's phenomenon and healthy controls (respectively 230 (155-387), 182 (101-284), and 207 (109-291) pg/mg, p = 0.006). In patients suffering from systemic sclerosis, there was a significant inverse correlation between F2-isoprostanes and postocclusive hyperemia, expressed as raw data (R = -0.45, p = 0.007) or as an increase over baseline (R = -0.28, p = 0.04). Conversely, no correlation was found with the nitroglycerin response. In conclusion, we provide evidence that there is an inverse correlation between postocclusive hyperemia and urinary F2-isoprostane levels in patients suffering from systemic sclerosis. Whether oxygen free radicals initiate the vascular dysfunction or whether there is an initial trigger that initiates both processes will need to be further clarified in future studies.     

Regulation of [3H]D-Aspartate Release by the 5-F2t-Isoprostane and Its 5-Epimer in Isolated Bovine Retina

We have evidence that 15-F?-isoprostanes (15-F?-IsoPs) regulate excitatory neurotransmitter release in ocular tissues. Although 5-F?-IsoPs are abundantly produced in mammals, their pharmacological actions on neurotransmitter release remain unknown. In the present study, we compared the effect of the 5-F?-IsoP epimer pair, 5-F(2t)-IsoP (C5-OH in β-position) and 5-epi-5-F(2t)-IsoP (C5-OH in α-position), on K?-evoked [3H]D-aspartate release in isolated bovine retina. We further examined the role of prostanoid receptors on the inhibitory action of 5-epi-5-F(2t)-IsoP on [3H]D-aspartate overflow. Isolated bovine retina were prepared for studies of K?-evoked release of [3H]D-aspartate using the superfusion method. 5-epi-5-F(2t)-IsoP (0.01 nM to 1 μM), attenuated K?-evoked [3H]D-aspartate release in a concentration-dependent manner, with the inhibitory effect of 26.9% (P < 0.001; IC?? = 0.2 μM) being achieved at 1 μM concentration. Its 5-(S)-OH-epimer, 5-F(2t)-IsoP (0.1 nM-1 μM), exhibited an inhibitory biphasic action, yielding a maximal response of 35.7% (P < 0.001) at 10 nM concentration of the drug (IC?? value of 3 nM). Although the prostanoid-receptor antagonists, AH 6809 (10 μM; EP???/DP) and BAY-u3405 (10 μM; DP/Tx) exhibited no effect on 5-epi-5-F(2t)-IsoP (10 nM-1 μM)-mediated inhibition, SC-19220 (1 μM; EP?) completely reversed 5-epi-5-F(2t)-IsoP (0.1 μM and 1 μM)-induced attenuation of K?-evoked [3H]D-aspartate release. Similarly, both SC-51322 (10 μM; EP? and AH 23848 (1 μM; EP?) reversed the inhibitory action elicited by 5-epi-5-F(2t)-IsoP (0.1 μM) on the neurotransmitter release. We conclude that the 5-F?-IsoP epimer pair, 5-F(2t)-IsoP and 5-epi-5-F(2t)-IsoP, attenuate K?-induced [3H]D-aspartate release in isolated bovine retina presumably via prostanoid receptor dependent mechanisms. The trans-orientation of the allylic hydroxyl group at position C5 accounts for the apparent biphasic response exhibited by 5-F(2t)-IsoP on excitatory neurotransmitter release.     

Social suppression of ovarian cyclicity in captive and wild colonies of naked mole-rats, Heterocephalus glaber

To investigate the endocrine cause of reproductive suppression in nonbreeding female naked mole-rats, animals from 35 colonies were studied in captivity. Urinary and plasma progesterone concentrations were elevated in pregnant females (urine: 10.0-148.4 ng/mg Cr, 27 samples from 8 females; plasma: 3.6-30.0 ng/ml, 5 samples from 5 females; Days 21-40 of pregnancy) and cyclic breeding females (urine: 0.5-97.8 ng/mg Cr, 146 samples from 7 females; plasma: less than 1.0-35.4 ng/ml, 25 samples from 7 females). The latter group showed cyclic patterns of urinary progesterone, indicating a mean ovarian cycle length of 34.4 +/- 1.6 days (mean +/- s.e.m.) with a follicular phase of 6.0 +/- 0.6 days and a luteal phase of 27.5 +/- 1.3 days (19 cycles from 9 breeding females). In non-breeding females urinary and plasma progesterone values were undetectable (urine: less than 0.5 ng/mg Cr, 232 samples from 64 females; plasma: less than 1.0 ng/ml, 7 samples from 6 females). Breeding females had higher (P less than 0.001) plasma LH concentrations (3.0 +/- 0.2 mi.u./ml, 73 samples from 24 females) than did non-breeding females (1.6 +/- 0.1 mi.u./ml, 57 samples from 44 females). Urinary and plasma progesterone concentrations in non-breeding females from wild colonies situated near Mtito Andei, Kenya, were either below the assay sensitivity limit (urine: less than 0.5 ng/mg Cr, 11 females from 2 colonies; plasma: less than 1.0 ng/ml, 25 females from 4 colonies), or very low (plasma: 1.6 +/- 0.6 ng/ml, 15 females from 4 colonies). In captivity, non-breeding females removed from their colonies (i.e. the dominant breeding female) and either paired directly with a non-breeding male (N = 2), or removed and housed singly for 6 weeks before pairing with a non-breeding male (N = 5) may develop a perforate vagina for the first time in as little as 7 days. Urinary progesterone concentrations rose above 2.0 ng/mg Cr (indicative of a luteal phase) for the first time 8.0 +/- 1.9 days after being separated. These results suggest that ovulation is suppressed in subordinate non-breeding female naked mole-rats in captive and wild colonies, and show that plasma LH concentrations are significantly lower in these non-breeding females. This reproductive block in non-breeding females is readily reversible if the social factors suppressing reproduction are removed.     

Detrimental effects of environmental tobacco smoke in relation to asthma severity

Background

Environmental tobacco smoke (ETS) has adverse effects on the health of asthmatics, however the harmful consequences of ETS in relation to asthma severity are unknown.

Methods

In a multicenter study of severe asthma, we assessed the impact of ETS exposure on morbidity, health care utilization and lung functions; and activity of systemic superoxide dismutase (SOD), a potential oxidative target of ETS that is negatively associated with asthma severity.

Findings

From 2002–2006, 654 asthmatics (non-severe 366, severe 288) were enrolled, among whom 109 non-severe and 67 severe asthmatics were routinely exposed to ETS as ascertained by history and validated by urine cotinine levels. ETS-exposure was associated with lower quality of life scores; greater rescue inhaler use; lower lung function; greater bronchodilator responsiveness; and greater risk for emergency room visits, hospitalization and intensive care unit admission. ETS-exposure was associated with lower levels of serum SOD activity, particularly in asthmatic women of African heritage.

Interpretation

ETS-exposure of asthmatic individuals is associated with worse lung function, higher acuity of exacerbations, more health care utilization, and greater bronchial hyperreactivity. The association of diminished systemic SOD activity to ETS exposure provides for the first time a specific oxidant mechanism by which ETS may adversely affect patients with asthma.     

Cyclo-oxygenase-1 and -2 differently contribute to prostaglandin E2 synthesis and lipid peroxidation after in vivo activation of N-methyl-D-aspartate receptors in rat hippocampus

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-isoprostane 15-F(2t)-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites--produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,--was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F(2t)-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.     

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.