A mechanical task for measuring sign- and goal-tracking in humans: A proof-of-concept study.

Cue-based associative learning (i.e., Pavlovian conditioning) is a foundational component of behavior in almost all forms of animal life and may provide insight into individual differences in addiction liability. Cues can take on incentive-motivational properties (i.e., incentive salience) through Pavlovian learning. Extensive testing with non-human animals (primarily rats) has demonstrated significant variation among individuals in the behaviors this type of learning evokes. So-named "sign-trackers" and "goal-trackers" have been examined in many studies of non-human animals, but this work in humans is still a nascent area of research. In the present proof-of-concept study, we used a Pavlovian conditioned approach task to investigate human sign- and goal-tracking in emerging adults. Conditioned behaviors that developed over the course of the task were directed toward the reward-cue and toward the reward location. Participants' eye-gaze and behavior during the task were submitted to a latent profile analysis, which revealed three groups defined as sign-trackers (n = 10), goal-trackers (n = 4), and intermediate responders (n = 36). Impulsivity was a significant predictor of the sign-tracking group relative to the goal-tracking group. The present study provides preliminary evidence that a simple procedure can produce learned Pavlovian conditioned approach behavior in humans. Though further investigation is required, findings provide a promising step toward the long-term goal of translating important insights gleaned from basic research into treatment strategies that can be applied to clinical populations.

The minimum important difference for the Pelvic Organ Prolapse-Urinary Incontinence Sexual Function Questionnaire.

INTRODUCTION AND HYPOTHESIS: Although the Pelvic Organ Prolapse-Urinary Incontinence Sexual Function Questionnaire (PISQ) is widely used to assess sexual function in women, the minimum important difference (MID) (defined as the smallest difference in scores of a patient-reported outcome measure that is perceived by patients as beneficial or harmful and which would lead the clinician to consider a change in treatment) is not known. The objective was to estimate the MID for the PISQ. METHODS: Two study populations, one of women with overactive bladder (OAB) and urgency UI (UUI) treated with tolterodine in a placebo-controlled trial (cohort I), and one of women treated surgically for prolapse and/or UI (cohort II) were used. Cohort I anchors were the Overactive Bladder Questionnaire (OAB-q), the Patient Perception of Bladder Condition (PPBC), the Patient Perception of Treatment Benefit Questionnaire (PPTBQ), and the change in number of UUI episodes in bladder diaries. Distribution MIDs were also calculated. RESULTS: In the anchor-based analysis, the MID values for changes in PISQ total scores at 3 months in cohort I were 5 points using the UUI anchor (diary-dry women), 5 points using the PPBC anchor, 5 points with the PPTBQ, and 9 points with the OAB-q. In cohort II, the MID at week 12 in PISQ total scores was 7 points in women with improved IIQ-7 scores. The distribution-based MID in PISQ total scores was 5.3 points in cohort I and 5.8 points in cohort II. CONCLUSION: A reasonable estimate of MID for the PISQ total score is 6 points. Improvements that meet these thresholds may be considered clinically important.

Biomarker responses and disease susceptibility in juvenile rainbow trout Oncorhynchus mykiss fed a high molecular weight PAH mixture.

Juvenile rainbow trout were fed a diet containing an environmentally relevant mixture of 10 high molecular weight polycyclic aromatic hydrocarbons (PAHs) at a dose of 0.66 or 7.82 µg PAH · g fish(-1) · d(-1). At 3, 7, 14, and 28 d, biomarkers of aryl hydrocarbon receptor activation (AHR), hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity, and cytochrome P4501A (CYP1A)-associated staining increased 14- to 26-fold and 6- to 14-fold, respectively, in fish fed 7.82 µg PAH · g fish (-1) · d(-1). Cytochrome P4501A-associated staining increased 2- to 9-fold on days 3, 7, and 28 in fish fed 0.66 µg PAH · g fish(-1) · d(-1). Bile fluorescent aromatic compounds served as a biomarker of exposure and confirmed that PAH exposure was consistent over 50 d. DNA damage in blood cells, protein oxidation, and lipid peroxidation in the kidney were biomarkers of oxidative stress and all increased in fish fed 7.82 µg PAH · g fish(-1) · d(-1). Fish fed 0.66 µg PAH · g fish(-1) · d(-1) had elevated DNA damage in blood cells but increased protein oxidation or lipid peroxidation in the kidney were not observed. Challenge with Aeromonas salmonicida, at lethal concentration (LC) 20, decreased survival in fish previously fed either 0.66 µg PAH · g fish(-1) · d(-1) or 7.82 µg PAH · g fish(-1) · d(-1) relative to fish fed the control diet. In general, biomarkers of both AHR activation and oxidative stress peaked at 3 to 14 d then declined at 28 to 50 d of PAH exposure and an increase in susceptibility to disease was observed at 50 d. These results link PAH exposure to biomarker responses that may be useful as early indicators of population level responses, such as mortality resulting from an increase in disease susceptibility.

Low-Sodium DASH reduces oxidative stress and improves vascular function in salt-sensitive humans.

Salt induces oxidative stress in salt-sensitive (SS) animals and man. It is not known whether in SS subjects the low-sodium dietary approaches to stop hypertension (LS-DASH) reduces oxidative stress more than DASH, which is high in antioxidants. To assess the effects of DASH and LS-DASH on oxidative stress, 19 volunteers were studied after 3 weeks of a standardized usual low fruits and vegetables diet (ULFV), followed by 3 weeks on DASH (both diets approximately 120 mmol Na(+) per day), then 3 weeks on LS-DASH (60 mmol Na(+) per day). SS was defined as systolic blood pressure >or=5 mm Hg lower on LS-DASH than DASH. In SS subjects (N=9), systolic blood pressure was lower on LS-DASH (111.0+/-2.0 mm Hg) than DASH (118.0+/-2.2, P<0.01) and ULFV (122.3+/-2.7, P=0.002). In salt-resistant (SR) volunteers (N=10), systolic blood pressure was lower on DASH (113.0+/-1.6) than ULFV (119.0+/-1.8, P<0.05) but not LS-DASH (115.7+/-1.8). Urine F2-isoprostanes, a marker of oxidative stress, were lower in SS subjects on LS-DASH (1.69+/-0.24) than ULFV (3.09+/-0.50, P<0.05) and marginally lower than DASH (2.46+/-0.44, P<0.20). F2-isoprostanes were not different among the three diets in SR volunteers (2.18+/-0.29, 2.06+/-0.29, 2.27+/-0.53, respectively). Aortic augmentation index, a measure of vascular stiffness, was lower in SS subjects on LS-DASH than either DASH or ULFV, and lower on DASH than ULFV in SR volunteers. In SS but not SR subjects, LS-DASH is associated with lower values for F2-isoprostanes and the aortic augmentation index. The results suggest that LS-DASH decreases oxidative stress, improves vascular function and lowers blood pressure in SS but not SR volunteers.

Effects of flexible-dose fesoterodine on overactive bladder symptoms and treatment satisfaction: an open-label study.

AIMS: To evaluate the efficacy and tolerability of flexible-dose fesoterodine in subjects with overactive bladder (OAB) who were dissatisfied with previous tolterodine treatment. METHODS: This was a 12-week, open-label, flexible-dose study of adults with OAB (> or = 8 micturitions and > or = 3 urgency episodes per 24 h) who had been treated with tolterodine (immediate- or extended-release) for OAB within 2 years of screening and reported dissatisfaction with tolterodine treatment. Subjects received fesoterodine 4 mg once daily for 4 weeks; thereafter, daily dosage was maintained at 4 mg or increased to 8 mg based on the subject's and physician's subjective assessment of efficacy and tolerability. Subjects completed 5-day diaries, the Patient Perception of Bladder Condition (PPBC) and the Overactive Bladder Questionnaire (OAB-q) at baseline and week 12 and rated treatment satisfaction at week 12 using the Treatment Satisfaction Question (TSQ). Safety and tolerability were assessed. RESULTS: Among 516 subjects treated, approximately 50% opted for dose escalation to 8 mg at week 4. Significant improvements from baseline to week 12 were observed in micturitions, urgency urinary incontinence episodes, micturition-related urgency episodes and severe micturition-related urgency episodes per 24 h (all p < 0.0001). Approximately 80% of subjects who responded to the TSQ at week 12 reported satisfaction with treatment; 38% reported being very satisfied. Using the PPBC, 83% of subjects reported improvement at week 12 with 59% reporting improvement > or = 2 points. Significant improvements from baseline (p < 0.0001) exceeding the minimally important difference (10 points) were observed in OAB-q Symptom Bother and Health-Related Quality of Life (HRQL) scales and all four HRQL domains. Dry mouth (23%) and constipation (5%) were the most common adverse events; no safety issues were identified. CONCLUSION: Flexible-dose fesoterodine significantly improved OAB symptoms, HRQL, and rates of treatment satisfaction and was well tolerated in subjects with OAB who were dissatisfied with prior tolterodine therapy.

Why the United States still needs improved dietary supplement regulation and oversight.

It has been 3 years since the American Society for Clinical Pharmacology and Therapeutics (ASCPT) issued a position statement regarding dietary supplement safety and regulation. I was the Chair of the ASCPT task force charged with issuing the statement. At the time, after careful review of available data, the other members and I concluded that dietary supplement legislation in the United States was lacking and that enhanced oversight was essential to increase the safety of these products for the American consumer.

Effect of pharmacological lowering of plasma urate on exercise-induced oxidative stress.

Urate is a metabolic end product of purine metabolism that contributes about 66% of the antioxidant capacity of plasma. The objective of this study was to evaluate the importance of plasma urate as an antioxidant using pharmacological lowering and examining the impact on plasma antioxidant capacity and oxidative stress after intense exercise. Fifteen subjects ran for 45 min at approximately 80% VO2 max under the influence of probenecid (1 g/d) (PRO) or placebo (PLA) in a double-blind, crossover design. Blood samples obtained at baseline, pre-exercise, and immediately post-exercise were analyzed for F2-isoprostanes, lipid hydroperoxides (LHs), ferric-reducing ability of plasma (FRAP), urate, ascorbate (AA), and nitrite. A 2 (group)x2 (time) repeated-measures analysis of variance (ANOVA), one-way ANOVA, Tukey-Kramer multiple comparison tests, and Student's t tests were used for statistical analysis. PRO exhibited lowered urate and FRAP compared with baseline (p

Oxidative stress in systemic lupus erythematosus: relationship to disease activity and symptoms.

Oxidative stress may play a role in the pathogenesis of systemic lupus erythematosus (SLE). We examined the hypothesis that oxidative stress was associated with indices of lupus disease activity and severity of symptoms. Urinary F2 isoprostane excretion, a validated marker of oxidative stress, was measured in 95 patients with SLE and 103 healthy controls. Outcome measures included SLEDAI and SLICC scores, the modified health assessment questionnaire, the fatigue severity scale (FSS), and visual analogue scales (VAS) for fatigue, pain and overall disease activity. F2 isoprostane excretion was compared in patients and controls, and its relationship with clinical variables in SLE examined. F2 isoprostane excretion did not differ significantly among patients with lupus (2.7 +/- 2.3 ng/mg Cr) and control subjects (2.2 +/- 1.4 ng/mg Cr) (P = 0.70). In patients with lupus, F2 isoprostane concentrations were independently associated with higher patient reported disease activity (VAS) (OR = 1.52, P = 0.01), fatigue (FSS, OR = 1.52, P = 0.03) and lower quality of life (OR = 0.73, P = 0.05), but not with objective markers or inflammation or disease activity. In conclusion, F2 isoprostane excretion is associated with patient-reported symptoms in SLE but not with measures of inflammation, SLEDAI or SLICC. Oxidative stress may contribute to debilitating symptoms such as fatigue in SLE.

Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.

Mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and have been hypothesized to contribute to aging. We show that mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase g (POLG) accumulate mtDNA mutations and display features of accelerated aging. Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of apoptotic markers, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice. Thus, accumulation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging.

Hyperthermia increases exercise-induced oxidative stress.

The purpose of this investigation was to examine oxidative markers after exercise in a hyperthermic environment (35 degrees C, 70 % RH) (Hot) versus a neutral environment (25 degrees C, 40 % RH) (Con). Hyperthermia may exacerbate oxidative stress by uncoupling the mitochondrial respiratory chain or by inhibiting antioxidant defense mechanisms, but this has not been assessed in vivo. Six male subjects performed low-intensity exercise (50 % VO(2max)) on a treadmill in Hot until a core temperature of 39.5 degrees C was reached, and for an equivalent time in Con. Blood samples were drawn before and immediately after exercise and at 8 min and 15 min following exercise. Samples were analyzed for F2 isoprostanes (FIP), lipid hydroperoxides (LPO), and lactate. A 2 x 4 repeated measures ANOVA was used to test for treatment, time, and interaction effects for FIP, LPO, and lactate. Differences in VO(2) were tested with Student's t-test. Significance was set at p < 0.05. Oxygen consumption was not significantly different between Hot and Con. The pattern of change of FIP and lactate in Hot was significant versus exercise in Con. LPO was significantly elevated over time in both Hot and Con, but the pattern of change was not significantly different. Ending core temperatures and heart rates were significantly elevated in Hot versus Con. These data indicate that hyperthermia increases oxidative stress and selectively affects specific lipid markers, independent of oxygen consumption.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.

C18 hydroxy fatty acids as markers of lipid peroxidation ex vivo and in vivo.

Different C18 monohydroxy fatty acids (OHFAs) were evaluated for their usefulness as markers of plasma lipid peroxidation (unsaturated fatty acid oxidation) ex vivo and in vivo. First, plasma samples (n = 5) were exposed for 3 h to different radical fluxes ex vivo. The formation of OHFAs was assessed by using varying concentrations of Cu2+ ions and AAPH (2,2'-azobis(2-amidinopropane) hydrochloride) as radical flux initiators. Secondly, a cross-sectional study was carried out in 47 middle-aged men. In this study, plasma concentrations of different in vivo OHFAs were compared with other indices of lipid peroxidation. Under mild oxidation conditions (heparin plasma containing 4.2 or 8.3 mM AAPH), concentrations of all the measured OHFAs (8, 9, 10, 11, 12, 13, 15 and 16-OH acids) increased in an identical manner, but under highly oxidative conditions (heparin plasma containing 83 mM AAPH or 4.2 to 8.3 mM CuSO4) mainly 9 and 13-OHFAs were formed. In the cross-sectional study, plasma 11 and 13-OHFA levels were associated statistically significantly with plasma free F2alpha-isoprostanes, recognized index of in vivo lipid peroxidation (r = 0.305, p = 0.037 and r = 0.308, p = 0.035, respectively). In addition, 16-OHFA levels correlated with the ratio of electronegatively charged LDL to total LDL (r = 0.335, p = 0.021). With respect to the other OHFAs, 15-OHFA had no correlation with either other OHFAs or the reference substances used. In addition, occasionally there were contamination problems in the assessment of 12-OHFA. It is concluded that all of the measured C18 OHFAs can be used as indicators of plasma lipid peroxidation under mild oxidation conditions, though the 12 and 15-OHFAs may need to be used with some caution. Under high oxidation conditions, 9-and 13-OHFAs seem to be the most useful indices because of their high formation capacity.

Inducible nitric oxide synthase regulates production of isoprostanes in vivo during chlamydial genital infection in mice.

Urinary nitrite and F(2)-isoprostanes, an index of oxidant stress, were elevated during chlamydial genital infection of mice. Enhancement of urinary nitrite and F(2)-isoprostanes was observed in phagocyte oxidase-deficient mice. Inhibition of inducible nitric oxide synthase reduced isoprostane excretion. We conclude that nitrogen radicals induce F(2)-isoprostane production and excretion during murine chlamydial genital infection.

Immune and oxidative changes during and following the Western States Endurance Run.

Changes in immune and oxidative stress parameters were measured in ultramarathon runners competing in the 160-km Western States Endurance Run. Forty-five runners agreed to provide blood and saliva samples the morning before the race event, at the 90-km aid station, and 5 - 10 min post-race. Upper respiratory tract infection (URTI) during the two-week period post-race was assessed retrospectively by telephone interviews. Forty subjects completed 90-km (race time, 13.1 +/- 0.3 h), and 31 completed the 160-km race event (27.0 +/- 0.4 h). The blood neutrophil and monocyte counts rose 249 % and 214 %, respectively, in the 31 finishers. Salivary IgA (sIgA) secretion rate decreased significantly from 508 +/- 40 micro g/min pre-race, to 287 +/- 39 micro g/min at 90-km, and 254 +/- 30 micro g/min post-race (50 % decrease). Significant increases were measured in cytokines at 90-km and post-race, with post-race IL-10 increasing 9.5-fold, IL-1ra 6.1-fold, IL-6 50.2-fold, and IL-8 2.5-fold over pre-race levels. Post-race indicators of oxidative stress, F (2)-isoprostane and lipid hydroperoxides, increased 33 % and 88 %, respectively. Pearson product-moment correlations revealed positive correlations at 90-km between F (2)-isoprostane and IL-6 (r = 0.31, p = 0.048), IL-10 (r = 0.31, p = 0.050), and IL-8 (r = 0.43, p = 0.005), but no other significant relationships between immune and oxidative stress indicators at 90-km and post-race. In the group of runners completing at least 90 km of the race, 26 % reported an URTI episode during the two-week period post-race. A low sIgA secretion rate at 90-km was the best predictor of post-race URTI (173 +/- 34 micro g/min in those who later acquired URTI compared to 325 +/- 40 micro g/min in those without URTI, p = 0.007). In conclusion, a modest correlation was found between cytokines and F (2)-isoprostane at 90-km when the greatest oxidative stress occurred, but no other significant correlations in immune and oxidative stress indicators during and following a 160-km ultramarathon race event were noted. About one in four ultramarathoners reported URTI during the two-week period post-race, and a low sIgA secretion rate mid-race best predicted URTI occurrence.

Plasma F2-isoprostane levels are elevated in chronic hemodialysis patients.

AIMS: Cardiovascular mortality has been reported to be 10- to 20-fold higher in chronic dialysis patients than in the age-matched general population. It has been suggested that increased oxidant stress and resulting vascular wall injury due to uremia and the hemodialysis procedure may be one of the mechanisms predisposing to these cardiovascular complications. Further, hemodialysis membrane bioincompatibility can contribute to increased oxidative stress and prevalence of inflammation. MATERIALS: We studied 18 chronic hemodialysis (CHD) patients (age 62.8 +/- 14.7 years, 39% male, 61% African-American, 44% insulin-dependent diabetic, 61% smokers, 61% with documented coronary artery disease) during hemodialysis with 2 membranes with different flux and complement activating properties. METHODS: We have measured free and phospholipid-bound F2-isoprostane (F2-IsoP) levels, a sensitive marker of oxidative stress, in CHD patients and compared them to levels in healthy subjects. We have also examined the acute effects of the hemodialysis procedure using both biocompatible and bioincompatible membranes on F2-IsoP levels. RESULTS: The results indicated that, compared to controls, both free (96.2 +/- 48.8 pg/ml versus 37.6 +/- 17.2 pg/ml) and bound F2-IsoP (220.4 +/- 154.8 pg/ml versus 146.8 +/- 58.4 pg/ml) levels were significantly higher (p < 0.05 for both). There was a statistically significant decrease in free F2-IsoP concentrations at 15 and 30 minutes of HD, which rebounded to baseline levels at the completion of the procedure. There were no significant differences in F2-IsoP concentrations between the 2 study dialyzers at any time point. Age, smoking status, diabetes mellitus and presence of cardiovascular disease were also not correlated with F2-IsoP levels in this patient population. There was a significant association between predialysis F2-IsoP and C-reactive protein concentrations. CONCLUSION: Using a sensitive and specific assay for the measurement of F2-IsoP, we demonstrated that CHD patients are under increased oxidative stress. During a single hemodialysis treatment, the hemodialysis membrane appears to have no discernable effect on oxidative stress status. Measurement of F2-isoprostanes may be a useful biomarker of oxidative stress status as well as in developing new therapeutic strategies to ameliorate inflammatory and oxidative injury in this patient population.

Products of the isoprostane pathway: unique bioactive compounds and markers of lipid peroxidation.

We previously reported the discovery of prostaglandin F2-like compounds (F2-isoprostanes) formed by nonenzymatic free-radical-induced peroxidation of arachidonic acid. Quantification of F2-isoprostanes has proven to be a major advance in assessing oxidative stress status in vivo. Central in the pathway of formation of isoprostanes are prostaglandin H2-like endoperoxides, which also undergo rearrangement in vivo to form E-ring, D-ring, and thromboxane-ring compounds. E2- and D2-isoprostanes also undergo dehydration in vivo to form reactive cyclopentenone A2- and J2-isoprostanes, which are susceptible to Michael addition reactions with thiols. Recently, we described the formation of highly reactive gamma-ketoaldehydes (now termed isoketals) as products of isoprostane endoperoxide rearrangement which readily adduct to lysine residues on proteins and induce cross-links at rates that far exceed other aldehyde products of lipid peroxidation. Isoprostane-like compounds (neuroprostanes) and isoketal-like compounds (neuroketals) are formed from oxidation of docosahexaenoic acid, which is enriched in the brain, and measurement of neuroprostanes may provide a unique marker of oxidative neuronal injury.

In-vivo effects of Glu298Asp endothelial nitric oxide synthase polymorphism.

Endothelial nitric oxide synthase catalyses the formation of the vasodilator nitric oxide, a major regulator of vascular tone. The Asp298 polymorphism of the nitric oxide synthase gene is associated with altered function and expression of the enzyme in vitro and myocardial infarction and coronary artery spasm in vivo. We examined the effect of the Glu298Asp polymorphism on: (1) local vascular responses to phenylephrine, acetylcholine, glyceryl trinitrate and prostaglandin E1 in the dorsal hand vein; (2) changes in forearm blood flow during mental stress, a measure of nitric oxide-mediated effect on resistance vessels; (3) excretion of urinary nitrite/nitrate as a measure of total body nitric oxide production; and (4) F2-isoprostane metabolite, a measure of oxidative stress, in healthy Glu298 (n = 12) and Asp298 (n = 13) homozygotes. There were no significant differences in acetylcholine dose responses (P = 0.29) in Glu298 and Asp298 homozygotes. Responses to glyceryl trinitrate, prostaglandin E1 and the alpha-adrenergic agonist phenylephrine did not differ by genotype. Forearm blood flow was similar at rest and increased significantly (from 7.5 ml/min/100 ml to 12.2 ml/min/100 ml; P = 0.003), but similarly (P = 0.2), during mental stress in both genotypes. Asp298 homozygotes excreted significantly less nitrate/nitrite than Glu298 homozygotes (nitrate + nitrite/creatinine ratio 0.05 +/- 0.01 vs. 0.09 +/- 0.01, respectively; P < 0.005). Urinary F2-isoprostane metabolite excretion did not differ (Glu298, 2.04 +/- 0.25 ng/mg creatinine; Asp298, 1.85 +/- 0.37 ng/mg creatinine; P = 0.7). We conclude that in healthy volunteers the Glu298Asp polymorphism affects endogenous nitric oxide production without affecting nitric oxide-mediated vascular responses. This polymorphism may only have clinical significance in the presence of endothelial dysfunction.

Mechanisms of ascorbic acid recycling in human erythrocytes.

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75-90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH.

Improved assay for the quantification of the major urinary metabolite of the isoprostane 15-F(2t)-Isoprostane (8-iso-PGF(2alpha)) by a stable isotope dilution mass spectrometric assay.

BACKGROUND: The F(2)-isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical catalyzed peroxidation of arachidonic acid. One IsoP, 15-F(2t)-IsoP (8-iso-PGF(2alpha)), has been shown to be formed in abundance in vivo and to exert potent biological activity. METHODS: As a means to assess the endogenous production of this compound, we previously developed a method to quantify the major urinary metabolite of 15-F(2t)-IsoP, 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha), 15-F(2t)-IsoP-M ), by gas chromotography (GC)/negative ion chemical ionization mass spectrometry (MS) employing stable isotope dilution methodology. While useful, we found that the assay occasionally suffered from the presence of impurities that co-elute on GC with 15-F(2t)-IsoP-M, making the measurement of this compound difficult. We now report a modified assay for the quantification of 15-F(2t)-IsoP-M employing GC/MS that alleviates this problem. RESULTS: Precision of the assay is +/-7% and the accuracy is 96%. The lower limit of sensitivity is approximately 8 pg. Normal concentrations of this metabolite in urine were found to be 0.46+/-0.09 ng/mg creatinine (mean+/-1 S.D.) Urinary excretion of 15-F(2t)-IsoP-M is markedly altered in situations associated with increased or decreased oxidant stress in vivo. CONCLUSIONS: This assay provided a sensitive and accurate method to assess endogenous IsoP generation and can be used to further explore the role of oxidant injury in human disease.