Jarosław Markowski

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.

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.

The Assessment Of The Risk Of Allergenicity Of ‘Sabina’ And ‘Debreceni Bötermö’ Sour Cherry Cvs (Prunus Cerasus L.) In A Guinea Pig Model

Abstract The allergic reactions to fruits are lesser known among food sensitivities. The most common fruits belonging to the Rosaceae family that might cause allergic reactions are apples, pears and peaches. However, little is known about the potential allergic reactions caused by another member of the Rosaceae, the cherry. The aim of this study was to assess the risk of any allergic reaction or food hypersensitivity resulting from topical application and chronic oral administration of cherry fruits. The cherry fruits ‘Sabina’ cv. were produced in the orchard in Dąbrowice according to the principles of integrated (IFP) and organic (OR) productions. Fruits of ‘Debreceni Bötermö’ cv. were produced in Dąbrowice (IFP), and in the orchard in Nowy Dwór (OR). The experiments were performed on 65 outbred young, adult, white albinotic guinea pigs (Dankin Hartley). Three procedures were applied: I. Guinea-Pig Maximization Test (GPMT); II. Chronic administration of fruits and III. Skin prick (Dreborg) test. The skin reactions based on GPMT or Dreborg tests revealed no differences between the two cherry cultivars ‘Sabina’ and ‘Debreceni’ obtained from integrated or organic production. Similarly, it was not observed of any effect of cultivars of cherries nor the type of fruits production on the guinea pig skin reaction as a result of chronic feeding with fruits.