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

• Published in: Clinical chemistry and laboratory medicine Vol. 46; no. 3; pp. 342 - 349
• Main Authors: Chrzczanowicz, Jacek, Gawron, Anna, Zwolinska, Anna, de Graft-Johnson, Jeffrey, Krajewski, Wojciech, Krol, Maciej, Markowski, Jaroslaw, Kostka, Tomasz, Nowak, Dariusz
• Format: Journal Article
• Language: English
• Published: Berlin Walter de Gruyter 01.01.2008; New York, NY
• Subjects: 2-diphenyl-1-picryl-hydrazyl (DPPH) radical; Acetonitriles - chemistry; Adult; Albumins - metabolism; Antioxidants - administration & dosage; Antioxidants - pharmacology; Beverages; Biological and medical sciences; Biphenyl Compounds; Chemical Precipitation; DPPH test; Female; Ferric Compounds - metabolism; ferric reducing ability of serum; Food; Free Radical Scavengers - blood; Free Radical Scavengers - metabolism; Free Radical Scavengers - pharmacology; General aspects; Humans; Investigative techniques, diagnostic techniques (general aspects); Linear Models; Male; Malus; Medical sciences; Middle Aged; Oxidation-Reduction; Picrates - blood; Picrates - metabolism; Picrates - pharmacology; Reproducibility of Results; Sensitivity and Specificity; Specimen Handling; Temperature; total serum antioxidant activity; Human; Nutrition; ferric reducing ability of serum; Exploration; Method; Antioxidant; total serum antioxidant activity; Biological activity; Feeding; Medicine; Diet; Clinical biology; Serum; 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical; DPPH test
• ISSN: 1434-6621; 1437-4331
• DOI: 10.1515/CCLM.2008.062

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.