Increased Circulating H3 Histone in Response to Repeated Bouts of Exercise Does Not Associate with Parallel Alterations of Cell-Free DNA.

Numerous studies have shown that cf nDNA significantly rises in stress caused by exercise. However, during nuclear decondensation, released DNA is followed by histones. Histones are also a common disease marker. After PAD4 mediated hypercitrullination extracellular H3Cit exhibits high toxicity contributing to tissue damage which, in cases of systemic inflammation, may lead to multiorgan failure and finally to death. We tested whether circulating histones rise in response to strenuous exercise. Eleven average-trained men performed three treadmill exercise tests to exhaustion at speed corresponding to 70% VO2max separated by 72 h of resting. Blood was collected before and just after each bout of exercise and plasma proteins were measured using enzyme-linked immunosorbent assay, whereas platelet activity was estimated with Light Transmission Aggregometry. Both, circulating histones and PAD4 raised in response to exercise. Plasma citrullinated histones increased from 3.1 ng/mL to 5.96 ng/mL (p = 0.0059), from 3.65 ng/mL to 6.37 ng/mL (p = 0.02), and from 3.86 ng/mL to 4.75 ng/mL (p = 0.033) after the first, second, and third treadmill run, respectively. However despite the parallel increase, no significant correlation between citrullinated histone and aggregation or cell-free nDNA was found. Furthermore, positive correlations of cf nDNA with aggregation and PAD4, lactate with aggregation, and lactate with citrullinated histone have been observed.

The Presence of Caffeic Acid in Cerebrospinal Fluid: Evidence That Dietary Polyphenols Can Cross the Blood-Brain Barrier in Humans.

Epidemiological data indicate that a diet rich in plant polyphenols has a positive effect on brain functions, improving memory and cognition in humans. Direct activity of ingested phenolics on brain neurons may be one of plausible mechanisms explaining these data. This also suggests that some phenolics can cross the blood-brain barrier and be present in the brain or cerebrospinal fluid. We measured 12 phenolics (a combination of the solid-phase extraction technique with high-performance liquid chromatography) in cerebrospinal fluid and matched plasma samples from 28 patients undergoing diagnostic lumbar puncture due to neurological disorders. Homovanillic acid, 3-hydroxyphenyl acetic acid and caffeic acid were detectable in cerebrospinal fluid reaching concentrations (median; interquartile range) 0.18; 0.14 µmol/L, 4.35; 7.36 µmol/L and 0.02; 0.01 µmol/L, respectively. Plasma concentrations of caffeic acid (0.03; 0.01 µmol/L) did not correlate with those in cerebrospinal fluid (ρ = -0.109, p = 0.58). Because food (fruits and vegetables) is the only source of caffeic acid in human body fluids, our results indicate that the same dietary phenolics can cross blood-brain barrier in humans, and that transportation of caffeic acid through this barrier is not the result of simple or facilitated diffusion.

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

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.

Hemodialysis Decreases the Concentration of Accumulated Plant Phenols in the Plasma of Patients on Maintenance Dialysis: Influence of Residual Renal Function.

Plant phenols may accumulate in end-stage kidney disease. The effect of hemodialysis on their plasma concentration remains poorly determined. Contingent on concentration, health-promoting or noxious effects occur; therefore, we assessed plasma concentration in hemodialyzed patients. In total, 21 maintenance hemodialyzed patients with diuresis < 500 mL per day (with oliguria), nine hemodialyzed patients with diuresis ≥ 500 mL per day (without oliguria) and 31 healthy volunteers were included. Nine phenolic acids were identified with high-performance liquid chromatography and total polyphenol concentration was determined with the Folin-Ciocalteu method in pre- or post-hemodialysis plasma and pre- or intra-hemodialysis dialysate. The concentration of total polyphenols was 27% higher in pre-hemodialysis plasma than in that of controls (0.95 ± 0.18 mmol/L [P < 0.0001]). The concentration of total polyphenols was higher in patients with oliguria (1.01 ± 0.17) than in those without (0.84 ± 0.13 mmol/L), despite the former having more intense hemodialysis (Kt/V 1.29 ± 0.31 and 0.77 ± 0.25, respectively). Pre-hemodialysis phenolic acid concentration in patients undergoing dialysis exceeded reference values by 3 to 34 times (3-hydroxyphenylacetic acid and vanillic acid, respectively), from 0.69 (dihydrocaffeic acid) to 169.3 µmol/L (hippuric acid). The concentration of six phenolic acids (3-hydroxyhippuric, caffeic, dihydrocaffeic, hippuric, homovanillic, and vanillic acid) was 1.1 (homovanillic) to 11.3 (3-hydroxyhippuric) times higher in patients with oliguria than in those without. 4-hydroxyhippuric acid occurred more in the plasma of patients with oliguria than in those without oliguria. A single hemodialysis session decreased total polyphenol concentration by 16% and phenolic acids from 30% (caffeic) to 58% (vanillic and 3-hydroxyphenylacetic acid) and these compounds appeared in the dialysate. The percentage decrease (Δ%) of creatinine concentration correlated with the Δ% of total polyphenols and five phenolic acids (3-hydroxyphenylacetic, dihydrocaffeic, hippuric, homovanillic, and vanillic acid). Urea Δ% and Kt/V correlated only with the Δ% of homovanilic acid. The results demonstrate that phenols accumulate variably in hemodialyzed patients and are differently eliminated during hemodialysis. Residual renal function ensures a lower concentration of plasma phenols.

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/m(2)) 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 (Æ¿ from 0.33 to 0.46 at the first 7 time points, p < 0.05). This association disappeared at the end of study (Æ¿ = 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.

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.

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.

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.

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.

Low-temperature molecular dynamics simulations of horse heart cytochrome c and comparison with inelastic neutron scattering data.

Molecular dynamics (MD) simulation combined with inelastic neutron scattering can provide information about the thermal dynamics of proteins, especially the low-frequency vibrational modes responsible for large movement of some parts of protein molecules. We performed several 30-ns MD simulations of cytochrome c (Cyt c) in a water box for temperatures ranging from 110 to 300 K and compared the results with those from experimental inelastic neutron scattering. The low-frequency vibrational modes were obtained via dynamic structure factors, S(Q, ω), obtained both from inelastic neutron scattering experiments and calculated from MD simulations for Cyt c in the same range of temperatures. The well known thermal transition in structural movements of Cyt c is clearly seen in MD simulations; it is, however, confined to unstructured fragments of loops Ω1 and Ω2; movement of structured loop Ω3 and both helical ends of the protein is resistant to thermal disturbance. Calculated and experimental S(Q, ω) plots are in qualitative agreement for low temperatures whereas above 200 K a boson peak vanishes from the calculated plots. This may be a result of loss of crystal structure by the protein-water system compared with the protein crystal.

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 driver's ability to perform complex sensory-motor activities? We compared a subject's 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 subject's 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.

[Comparison of local and systemic inflammatory markers in patients with community-acquired pneumonia and pneumonia coexisting with lung cancer]. / Porównanie wybranych parametrów lokalnego i systemowego stanu zapalnego u chorych na pozaszpitalne zapalenie pluc i zapalenie pluc wspólistniejace z rakiem pluca.

BACKGROUND: The aim of the study was to compare the local and systemic markers of inflammatory processes in patients with community-acquired pneumonia (CAP) and in those with pneumonia coexisting with lung cancer. MATERIAL AND METHODS: Seventeen patients with community-acquired pneumonia (group I), 14 patients with pneumonia and lung cancer (group II), and 24 patients with lung cancer (group III) were enrolled into the study. Sixteen healthy smokers served as a control group (group IV). Concentration of hydrogen peroxide (H(2)O(2)), vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-α) were measured in exhaled breath condensate (EBC). The levels of VEGF and TNF-α were also measured in serum. RESULTS: The concentrations of VEGF (317.83 ± 77.78) and TNF-α (1.98 ± 0.13) in EBC were significantly higher in patients with pneumonia and lung cancer as compared to patients with community-acquired pneumonia (VEGF 30.20 ± 6.56; TNF-α 0.31 ± 0.05). Also the level of H(2)O(2) (0.96 ± 0.16) in EBC in patients with pneumonia and lung cancer was elevated in comparison to patients with CAP (0.66 ± 0.09), however the difference was not statistically significant (p 〉 0.05). The serum concentrations of both studied cytokines were significantly higher in patients with pneumonia (VEGF 1112.62 ± ± 244.38 and TNF-α 2.6 ± 0.48) than in those with pneumonia and lung cancer (VEGF 392.9 ± 78.2; TNF-α 1.6 ± 0.2). CONCLUSIONS: Patients with pneumonia and lung cancer exhibited higher levels of oxidative stress and local inflammatory reactions than those with pneumonia. However, inflammatory markers in serum were significantly lower in patients with pneumonia and lung cancer as compared to those with CAP.

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.

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-microl 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 Fe(3 +). 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 micromol/l. No differences were found between FRAT of men and women. Only donor age correlated with FRAT (rho = 0.40, p < 0.01). Subjects 59 yrs (179.4 +/- 95.4 versus 237.2 +/- 151.7 micromol/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.

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

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 microL (r=0.99, p<0.01). With 25 microL-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 degrees C. Mean values of DPPH radical scavenging activity and FRAP obtained in human serum were 11.2+/-3.3% and 382.0+/-88.1 micromol/L, respectively. A positive significant linear correlation was observed between these two methods (r=0.42, p<0.01). Serum supplementation with 50 micromol/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.