Agata Sarniak

Apocynin reduces reactive oxygen species concentrations in exhaled breath condensate in asthmatics

ABSTRACT Asthma is an inflammatory airway disease, and oxidative stress was proven to be involved in its pathogenesis. Apocynin effectively inhibits the main source of reactive oxygen species (ROS)—nicotinamide adenine dinucleotide phosphate (NADPH) oxidase—by blocking its activation. The aim of this study was to investigate the effect of inhaled apocynin on ROS and RNS (reactive nitrogen species) concentration in 14 nonsmoking mild asthmatics. Effects of nebulized apocynin (0.5 mg/mL) were assessed in exhaled breath condensate (EBC) after 30, 60, and 120 minutes, and safety parameters have been analyzed. Apocynin significantly decreased H2O2 concentration in EBC in comparison with placebo after 60 and 120 minutes. Moreover, apocynin significantly reduced NO−2 concentration 30 and 60 minutes after nebulization and caused a significant decrease of NO−3 concentration in EBC 60 and 120 minutes after administration, comparing with placebo. No adverse events have been observed throughout the study. This research confirmed anti-inflammatory properties of nebulized apocynin, which might be an effective and safe drug in bronchial asthma.

Consumption of strawberries on a daily basis increases the non-urate 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity of fasting plasma in healthy subjects

Strawberries contain anthocyanins and ellagitanins which have antioxidant properties. We determined whether the consumption of strawberries increase the plasma antioxidant activity measured as the ability to decompose 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in healthy subjects. The study involved 10 volunteers (age 41 ± 6 years, body weight 74.4 ± 12.7 kg) that consumed 500 g of strawberries daily for 9 days and 7 matched controls. Fasting plasma and spot morning urine samples were collected at baseline, during fruit consumption and after a 6 day wash-out period. DPPH decomposition was measured in both deproteinized native plasma specimens and pretreated with uricase (non-urate plasma). Twelve phenolics were determined with HPLC. Strawberries had no effect on the antioxidant activity of native plasma and circulating phenolics. Non-urate plasma DPPH decomposition increased from 5.7 ± 0.6% to 6.6 ± 0.6%, 6.5 ± 1.0% and 6.3 ± 1.4% after 3, 6 and 9 days of supplementation, respectively. The wash-out period reversed this activity back to 5.7 ± 0.8% (p<0.01). Control subjects did not reveal any changes of plasma antioxidant activity. Significant increase in urinary urolithin A and 4-hydroxyhippuric (by 8.7- and 5.9-times after 6 days of supplementation with fruits) was noted. Strawberry consumption can increase the non-urate plasma antioxidant activity which, in turn, may decrease the risk of systemic oxidants overactivity.

Apocynin decreases hydrogen peroxide and nirtate concentrations in exhaled breath in healthy subjects

The imbalance between reactive oxygen species (ROS) synthesis and antioxidants might be involved in the pathogenesis of many inflammatory diseases. NADPH oxidase, an enzyme responsible for ROS production, may represent an attractive therapeutic target to inhibit, for the treatment of these diseases. Apocynin is an inhibitor of activation of NADPH oxidase complex present in the inflammatory cells. In double blind, placebo-controlled, cross-over study, we investigated the effect of nebulized apocynin on ROS synthesis in 10 nonsmoking healthy volunteers. Apocynin (6ml of 0.5mg/ml) was administered by nebulization and its effects on H(2)O(2), NO(2)(-) and NO(3)(-) generation were assessed after 30, 60 and 120min by collecting exhaled breath condensate (EBC) samples using an EcoScreen analyzer. Additionally, respiratory parameters have been evaluated, utilizing spirometry and DLCO. We also analyzed peripheral blood differential counts and NO(2)(-) serum level, cough scale control and blood pressure as safety parameters. Apocynin caused reduction of H(2)O(2) concentration in EBC as compared to placebo, after 60min. of inhalation (0.18microM vs. 0.31microM, p<0.05) as well as after 120min. (0.2microM vs. 0.31microM, p<0.05). Similarly, apocynin significantly decreased concentration of NO(3)(-) as compared to placebo, after 60 and 120min. (6.8microM vs. 14.4microM and 6.5microM vs. 14.9microM respectively, p<0.05). Apocynin was well tolerated and no adverse events have been observed throughout the study. Thus, as apocynin significantly influence ROS concentration, it might have also antiinflammatory properties. As it is safe, it may have a potential to become a drug in airway inflammatory diseases treatment.

Hydrogen peroxide and nitrite reduction in exhaled breath condensate of COPD patients

Chronic obstructive pulmonary disease (COPD) is predominantly the result of years of cigarette smoking. Increased oxidative stress in COPD derives from the increased burden of inhaled oxidants (cigarette smoke), air pollution and the increase in reactive oxygen and nitrogen species (ROS and RNS), generated by some inflammatory, immune, and structural airways cells. In view of the lack of therapy that might inhibit the progress of the disease, there is an urgent need for a successful therapeutic approach. Apocynin is a molecule inhibiting activation of NADPH oxidase - enzyme generating ROS and RNS precursor. Thus, our aim was to analyze apocynin influence on hydrogen peroxide and nitrite concentrations in EBC of COPD patients. Apocynin reduced concentration of H(2)O(2) in COPD patients 60 and 120 min after apocynin inhalation, in comparison to placebo (0.43 μM vs. 0.59 μM, and 0.4 μM vs. 0.59 μM respectively, p < 0.05). Moreover, apocynin decreased NO(2)(-) ions concentration in airways of COPD patients after apocynin nebulization (3.97 μM vs. 4.48 μM after 30 min, 3.82 μM vs. 4.48 μM after 60 min, and 3.76 μM vs. 4.48 μM after 30 min respectively, p < 0.05). No adverse effects have been observed. The results suggest that apocynin might be considered as anti-inflammatory agent, and, possibly used in therapy of COPD.

Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2−/NO3− in breath condensate

BACKGROUND Analysis of NO2-/NO3- in expired breath condensate (EBC) has been proposed as a marker of inflammation in various lung diseases. METHODS NO2- and total NO3-/NO2- concentrations were determined in EBC collected from healthy and asthmatic subjects. The NO3- was first reduced to NO2-, and total NO2- was detected by colorimetric Griess reaction. Two methods of NO3- reduction were compared. To reduce NO3-, cadmium (600 microl EBC-macromethod) and enzyme-NADPH-nitrate reductase (60 microl EBC-micromethod) were used. RESULTS Macromethod: Mean NO2- concentrations in EBC were 1.64 +/- 0.24 micromol/l in healthy subjects and 0.42 +/- 0.17 micromol/l in asthmatic patients. Mean total NO2-/NO3- levels were 3.64 +/- 0.43 micromol/l in healthy subjects and 3.27 +/- 0.34 micromol/l in asthmatic. Micromethod: NO2- level: 1.69 +/- 0.23 micromol/l in healthy subjects and 0.53 +/- 0.21 micromol/l in asthmatics. Total NO2-/NO3- levels: 3.56 +/- 0.37 micromol/l in healthy subjects and 3.57 +/- 1.17 micromol/l in asthmatics. Variability index was 27% and 6% for macro- and micromethod, respectively. Recovery of NO3- added to EBC was 100% for enzymatic and almost 88% for cadmium reduction. There was no correlation between total NO2-/NO3- levels determined by macro- and micromethod. CONCLUSIONS We recommend enzymatic reduction as a better method for NO3- determination in EBC.

Addition of Strawberries to the Usual Diet Decreases Resting Chemiluminescence of Fasting Blood in Healthy Subjects—Possible Health-Promoting Effect of These Fruits Consumption

Objective: Regular strawberry consumption augmented plasma antioxidant activity and decreased lipid peroxidation suggests preventive potential of these fruits against oxidative stress-dependent disorders. Blood phagocytes are important source of oxidants that may contribute to systemic oxidative stress. We examined the effect of strawberry consumption on the luminol enhanced whole blood chemiluminescence (LBCL) reflecting oxidants generation by circulating phagocytes in healthy subjects. Methods: Thirty-one healthy subjects (being on their usual diet) consumed 500 g of strawberry pulp daily (between 11.00–14.00) for 30 days (1st strawberry course) and after 10 day wash-out the cycle was repeated (2nd strawberry course). Fasting blood and spot morning urine samples were collected before and after each strawberry course for measuring resting and agonist (fMLP)-induced LBCL, various phenolics and plasma antioxidant activity. Twenty subjects served as a control in respect to LBCL changes over the study period. Results: Strawberry consumption decreased median resting LBCL and this effect was more evident after the 1st course (by 38.2%, p < 0.05) than after the the 2nd one (18.7%), while fMLP-induced LBCL was constant. No changes in LBCL were noted in controls. Strawberries increased fasting plasma levels of caffeic acid and homovanillic acid as well as urolithin A and 4-hydroxyhippuric acid in spot urine. Plasma antioxidant activity and the number of circulating phagocytes did not change over the study period. Resting LBCL correlated positively with the number of circulating polymorphonuclear leukocytes at all occasions and negative correlation with plasma 4-hydroxyhippuric acid was noted especially after the first strawberry course (r = −0.46, p < 0.05). Conclusions: The decrease in resting LBCL suggests that regular strawberry consumption may suppress baseline formation of oxidants by circulating phagocytes. This may decrease the risk of systemic imbalance between oxidants and anti-oxidants and be one of mechanisms of health-promoting effect of these fruits consumption.

Addition of strawberries to the usual diet increases postprandial but not fasting non-urate plasma antioxidant activity in healthy subjects

Strawberries can augment plasma antioxidant activity, but this may be confounded by selection of methods, time of blood sampling and concomitant dietary restrictions. We examined the effect of strawberry consumption on ferric reducing ability (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (DPPH-test) of native and non-urate plasma in healthy subjects on their usual diet. Eleven subjects consumed strawberries (500 g daily) for 9 days. Fasting and 3-h postprandial plasma and 24-h urine collection were obtained before, during and after strawberry course for FRAP, DPPH-test and polyphenols determination. Fifteen subjects served as a control in respect to plasma antioxidant activity changes and effect of 300 mg of oral ascorbate. First, 5th and 9th strawberry dose increased 3-h postprandial DPPH-test by 17.4, 17.6 and 12.6%, and FRAP by 15.5, 25.6 and 21.4% in comparison to fasting values in non-urate plasma (p<0.05). In native plasma only a trend was observed to higher postprandial values for both tests. Strawberries increased urinary urolithin A and 4-hydroxyhippuric acid whereas plasma polyphenols were stable. No changes of FRAP and DPPH-test were noted in controls and after ascorbate intake. Strawberries transiently increased non-urate plasma antioxidant activity but this cannot be attributed to direct antioxidant effect of polyphenols and ascorbate.

Strawberries Added to the Usual Diet Suppress Fasting Plasma Paraoxonase Activity and Have a Weak Transient Decreasing Effect on Cholesterol Levels in Healthy Nonobese Subjects

Objective: Strawberries can improve oxidants–antioxidants balance and reduce some cardiovascular risk factors in obese subjects. Paraoxonase-1 (PON-1) is a high-density lipoprotein–associated enzyme with antioxidant properties that can protect from coronary artery disease in humans. We examined the effect of strawberry consumption on plasma PON-1 activity and lipid profile in healthy nonobese subjects. Methods: Thirty-one subjects (body mass index [BMI] 24.4 ± 4.0 kg/m2) on their usual diet consumed 500 g of strawberry pulp daily for 30 days (first course) and after a 10-day washout the cycle was repeated (second course). Fasting blood and spot morning urine samples were collected before, during, and after each strawberry course (8 time points) for determination of paraoxonase and arylesterase PON-1 activities and lipid profile. Twenty subjects served as controls with respect to cholesterol and PON-1 activities changes over the study period. Results: Strawberries decreased mean plasma paraoxonase PON-1 activity and this effect was more evident after the second course (by 11.6%, p < 0.05) than after the first course (5.4%, p = 0.06), whereas arylesterase activity was constant. Strawberries altered total cholesterol levels (p < 0.05) with a tendency to transiently decrease it (by 5.1%) only after 15 days of the first course. Triglycerides and high- and low-density lipoprotein cholesterol did not change in response to fruit consumption. No changes in PON-1 activities and lipid profile were noted in controls. Paraoxonase correlated with arylesterase activity (ƿ from 0.33 to 0.46 at the first 7 time points, p < 0.05). This association disappeared at the end of study (ƿ = 0.07) when the strongest inhibition of paraoxonase was noted. Conclusions: Supplementation of the usual diet with strawberries decreased paraoxonase PON-1 activity and did not improve lipid profiles in healthy nonobese subjects. Further studies are necessary to establish the clinical significance of paraoxonase suppression and to define a group of healthy subjects who can benefit from strawberry consumption with respect to cholesterol levels.

Sour Cherries but Not Apples Added to the Regular Diet Decrease Resting and fMLP-Stimulated Chemiluminescence of Fasting Whole Blood in Healthy Subjects

ABSTRACT Objective: Berry fruits rich in anthocyanins have antioxidant and anti-inflammatory properties. Blood phagocytes are an important source of oxidants that contribute to inflammatory response and oxidative stress. We examined the effect of sour cherry consumption on luminol-enhanced whole blood chemiluminescence (LBCL) reflecting oxidants generation by circulating phagocytes in healthy subjects. Methods: Thirty-four and 29 healthy subjects (on a regular diet) consumed 500 g of sour cherries containing 346.5 mg of total anthocyanins or 500 g of anthocyanin-free apples everyday (between 1100 and 1400 hours) for 30 days. Twenty-four volunteers without any dietary intervention served as the control with respect to LBCL changes over the study period. Fasting blood and spot morning urine samples were collected before and after the fruit courses and after the 10-day wash-out period to measure resting and agonist (fMLP)-induced LBCL, blood cell count, concentration of various phenolics, and plasma antioxidant activity. Results: Sour cherries inhibited (p < 0.05) median resting LBCL (by 29.5% and 33.7%) and fMLP-LBCL (by 24.7% and 32.3%) after 30-day consumption and after 10-day wash-out, respectively. No changes in LBCL were noted in the apple consumers and controls. Increased urinary levels of chlorogenic, 4-hydroxyhippuric, and 3-hydroxyhippuric acids occasionally correlated negatively with resting and fMLP-LBCL in sour cherry consumers. Other measured variables did not change in all groups over the study period. Conclusions: The inhibition of resting and agonist-induced LBCL suggests that regular sour cherry consumption may suppress the formation of reactive oxygen species by circulating phagocytes and decrease the risk of systemic imbalance between oxidants and antioxidants. This may be attributed to the anthocyanins in sour cherry and be one of mechanisms of the health-promoting effects of consumption of anthocyanin-rich fruits.

Decreased integrity of exercise-induced plasma cell free nuclear DNA - negative association with the increased oxidants production by circulating phagocytes

Objective Strenuous exercise increases circulating cell free DNA (cf DNA) and stimulates blood phagocytes to generate more reactive oxygen species (ROS) which may induce DNA strand breaks. We tested whether: (A) elevated cf DNA in response to three repeated bouts of exhaustive exercise has decreased integrity; (B) each bout of exercise increases luminol enhanced whole blood chemiluminescence (LBCL) as a measure of ROS production by polymorphonuclear leukocytes; (C) there is an association between integrity of cf DNA and LBCL. Methods Eleven average-trained men performed three treadmill exercise tests to exhaustion at speed corresponding to 70% of their individual VO2 max separated by 72 hours of resting. Pre-and post-exercise concentrations and integrity of cf nuclear and mitochondrial DNA (cf n-DNA, cf mt-DNA) were determined with real-time PCR along with measurement of resting and fMLP-stimulated LBCL. Results Each bout increased concentrations of cf n-DNA by more than 10-times which was accompanied by about 2-times elevated post-exercise rLBCL and fMLP-LBCL (p<0.05). Post-exercise cf n-DNA integrity (integrity index, I206/78) decreased after the first (0.59±0.19 vs. 0.48±0.18, p<0.05) and second (0.53±0.14 vs. 0.44±0.17, p<0.05) bout of exercise. There were negative correlations between I206/78 and rLBCL > IJ–0.37, p<0.05) and I206/78 and fMLP-LBCL (ϱ =–T6*T4Ep<0.05) – analysis of pooled pre-and post-exercise data (n = 66). Although cf-mt DNA rose by about 2-times (p<0.05) after the second and third bout, its integrity (I218/97) did not alter in response to exercise. Conclusions Repeated bouts of exhaustive exercise caused increase in cf n-DNA with decreased integrity which correlated with increased ROS production by circulating polymorphonucler leukocytes. This suggests that oxidants may be involved in the release of cf n-DNA and cf n-DNA strand breaks in response to exhaustive exercise.