Anna Zwolinska

Simple method for determining human serum 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity : possible application in clinical studies on dietary antioxidants

Abstract Background: 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radical decomposition in alcohol solution is widely used, characterizing plant antioxidants that can rise in serum after fruit and vegetable intake. However, this test failed reproducible results with serum due to protein precipitation. We describe the application of serum deproteinization with acetonitrile relating to the DPPH test. Methods: Assay sensitivity, linearity, repeatability and storage effect were determined in serum samples deproteinized with an equal volume of acetonitrile. Associations between the DPPH test and the ferric reducing ability of serum (FRAP) method, measuring total antioxidant potential, were evaluated in sera from 78 healthy non-smoking men. The effect of a single ingestion of 1 L of cloudy apple juice on the serum DPPH radical scavenging activity in healthy volunteers was also investigated. Results: Assay linearity was within 5–25 μL (r=0.99, p<0.01). With 25 μL-deproteinized serum, coefficient of variation was 4.2% and detection limit was 0.5% of the initial amount of decomposed DPPH radical over 30 min incubation. There was no sera activity decrease over 14 days storage at –20°C. Mean values of DPPH radical scavenging activity and FRAP obtained in human serum were 11.2±3.3% and 382.0±88.1 μmol/L, respectively. A positive significant linear correlation was observed between these two methods (r=0.42, p<0.01). Serum supplementation with 50 μmol/L of catechin, gallic acid, ascorbic acid or uric acid enhanced DPPH test results. One brisk serving of 1 L of apple juice caused a significant increment of serum DPPH radical scavenging activity (1.9±1.9%, p<0.01) in 12 healthy subjects 1 h after juice ingestion. Conclusions: Applicability of the DPPH test to deproteinized serum with acetonitrile revealed numerous advantages, validating its practicability, simplicity and cost effectiveness as a tool in the estimation of antioxidant status in humans. Clin Chem Lab Med 2008;46:342–9.

Uric Acid but Not Apple Polyphenols Is Responsible for the Rise of Plasma Antioxidant Activity after Apple Juice Consumption in Healthy Subjects

Objective: To determine whether (1) rapid consumption of 1 L of apple juice increases blood antioxidant capacity, measured as ferric-reducing ability of plasma (FRAP) and serum 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, and (2) apple polyphenols or fructose-induced elevation of plasma uric acid contributes to post-juice increase of blood antioxidant activity. Methods: The study involved 12 (mean age 32 ± 5 years, mean body weight 73 ± 7 kg) healthy nonsmoking subjects. Tested subjects consumed 1 L of clear apple juice and then FRAP; serum DPPH-scavenging activity, serum uric acid, and total plasma phenolics and quercetin levels were measured just before juice ingestion and 1, 2.5, and 4 hours after ingestion. This was repeated 3 times with 4-day intervals, but volunteers drank either 1 L of clear apple juice without polyphenols (placebo), or 1 L of cloudy apple juice (positive control), or 1 L of water (negative control) at the time. All juices had similar content of sugars (i.e., saccharose, glucose, and fructose) and precisely defined composition of phenolics and antioxidant activity. Results: Consumption of all 3 juices transiently increased FRAP and serum DPPH-scavenging activity, with peak values at 1 hour post-juice ingestion. This was paralleled by the rise of serum uric acid, but no significant changes in plasma total phenolics and quercetin levels were observed after all dietary interventions. At the same time, no substantial differences were found between juices (especially between clear apple juice and clear apple juice without polyphenols) concerning the measured variables. A strong significant correlation was noted instead between serum uric acid and plasma antioxidant activity at all analyzed time points, before and after juice ingestion. Plasma total phenolics and quercetin levels were not associated with FRAP and serum DPPH radical-scavenging activity. Conclusions: We have demonstrated that rapid consumption of apple juice increased plasma antioxidant activity in healthy subjects; this was caused by the fructose-induced rise of serum uric acid levels, but was not due to the presence of antioxidant polyphenols in juice. Thus, short-term consumption of apple juice seems not to be the effective dietary intervention to augment plasma antioxidant activity due to the concomitant possibility for uric acid to be a risk factor for several diseases, as verified by other authors.

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.

Myocardial oxidative stress in patients with active infective endocarditis

BACKGROUND Infective endocarditis (IE) induces the rise of pro-inflammatory cytokines. Some of them can stimulate oxidants production in myocardium with subsequent peroxidative damage to various biomolecules. We compared indices of oxidative stress: H2O2, thiobarbituric acid-reactive substances (TBARs), thiols in myocardium specimens between patients with active IE and those with valvular heart disease (VHD) of rheumatic etiology who underwent surgical valve replacement. METHODS 17 left ventricle papillary muscle specimens and 28 specimens of auricle of the right heart were collected from 45IE patients, and 16 papillary muscle and 12 auricle specimens from 28 VHD patients, respectively. Patients groups had similar NYHA functional class and majority of echocardiographic indices of heart morphology. H2O2 and TBARs were determined fluorometrically in myocardium homogenates whereas thiols with photometric method. Between and within groups comparisons and mutual correlations between variables were analyzed. RESULTS H2O2 generation from all myocardium specimens and auricles was 2.14- and 2.59- times higher (p<0.001) in IE patients than in VHD group. Auricles had the highest H2O2 levels within IE group. TBARs were 10-times higher (p<0.05) in IE when compared to VHD group in auricles and papillary muscles. Thiols did not differ between groups. H2O2 positively correlated with TBARs and negatively with thiols in all IE myocardium specimens (r=0.31 and r=-0.46, p<0.05) and auricles (r=0.58 and r=-0.67, p<0.05), respectively. No such associations were noted in VHD specimens. CONCLUSIONS Active IE induces enhanced myocardial production of H2O2 and formation of TBARs which proves occurrence of oxidative stress in the heart.

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.

The sound of a mobile phone ringing affects the complex reaction time of its owner

Introduction Mobile phone conversation decreases the ability to concentrate and impairs the attention necessary to perform complex activities, such as driving a car. Does the ringing sound of a mobile phone affect the drivers ability to perform complex sensory-motor activities? We compared a subjects reaction time while performing a test either with a mobile phone ringing or without. Material and methods The examination was performed on a PC-based reaction time self-constructed system Reactor. The study group consisted of 42 healthy students. The protocol included instruction, control without phone and a proper session with subjects mobile phone ringing. The terms of the study were standardised. Results There were significant differences (p < 0.001) in reaction time in control (597 ms), mobile (633 ms) and instruction session (673 ms). The differences in female subpopulation were also significant (p < 0.01). Women revealed the longest reaction time in instruction session (707 ms), were significantly quicker in mobile (657 ms, p < 0.01) and in control session (612 ms, p < 0.001). In men, the significant difference was recorded only between instruction (622 ms) and control session (573 ms, p < 0.01). The other differences were not significant (p > 0.08). Men proofed to complete significantly quicker than women in instruction (p < 0.01) and in mobile session (p < 0.05). Differences amongst the genders in control session was not significant (p > 0.05). Conclusions The results obtained proofed the ringing of a phone exerts a significant influence on complex reaction time and quality of performed task.

Docosahexaenoic acid attenuates oxidative stress and protects human gingival fibroblasts against cytotoxicity induced by hydrogen peroxide and butyric acid.

OBJECTIVE The oxidative burst of the host cells associated with bacterial pathogen infection contributes to the destruction of periodontal tissue. The present study investigates the effect of docosahexaenoic acid (DHA) on human gingival fibroblast (HGF) viability and ROS generation. METHODS The cell viability by MTT assay, ROS level using H2DCF-DA probe, and protein thiol content were measured in HGFs after 24h preincubation with different concentrations of DHA followed by treatment with H2O2. The cell death rate was determined by Annexin V/propidium iodide staining, and mitochondrial membrane potential (ΔΨm) was examined by MitoTracker Red probe in H2O2- and butyric acid-treated HGFs. The fatty acid composition of plasma membranes after incubation with DHA was determined by gas chromatography mass spectrometry. RESULTS DHA preincubation in a dose-dependent manner increased the viability of HGFs exposed to H2O2 and decreased ROS generation compared to the control cells. In HGFs preincubated with 30μM DHA, the ΔΨm significantly increased in both H2O2- and butyric acid-treated cells. Moreover, incubation with DHA preserved the protein thiol level as effectively as N-acetylcysteine. Application of 50μM DHA increased the quantity of viable cells, decreased the number of necrotic cells after H2O2 treatment, and protected HGFs from apoptosis induced by butyric acid. DHA in the plasma membranes of these HGFs represented about 6% of the total amount of fatty acids. CONCLUSIONS These results demonstrate that enrichment of HGFs with DHA reduces ROS generation and enhances the mitochondrial membrane potential protecting the fibroblasts against cytotoxic factors.

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.

Ferric Reducing Ability of Tears in Healthy Subjects—Diurnal Variation and Dependence on Defined Demographic Data

Purpose: The assay of the ferric reducing ability of tears (FRAT) can be useful for monitoring ocular antioxidant capacity in clinical settings. FRAT diurnal variation was evaluated in healthy subjects and its relation with age, sex, body mass index, and ferric reducing ability of plasma (FRAP) were studied. Material and Methods: FRAT of 10-μ l tear samples collected with capillary tubes from 68 healthy subjects (age 10 to 92 yrs, 46 women, 22 men) were measured after 0, 3, 5, and 10 min of incubation with Fe3 +. FRAT diurnal variation was estimated in 11 subjects, with tear samples collected every 4 hr during the day. Comparison of FRAT versus FRAP was determined in 20 subjects. Results: Mean FRAT after 10-min incubation reached 207.7 ± 136.8 μ mol/l. No differences were found between FRAT of men and women. Only donor age correlated with FRAT (ρ = 0.40, p < 0.01). Subjects ≤ 59 years had lower FRAT levels than those age > 59 yrs (179.4 ± 95.4 versus 237.2 ± 151.7 μ mol/l, p < 0.01). For all investigated times of incubation, FRAT was approximately two times lower than FRAP (p < 0.01). FRAT revealed diurnal variation, with the highest value at 08:00 hours, upon awakening. Conclusion: The significance of age-matched controls and the specific time of the day for tears collection should be considered in the trial design and investigations with FRAT as a marker of the ocular antioxidant defense system.

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.