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.
Chrzczanowicz
Jacek
J
Department of Geriatrics, Medical University of Lodz, Lodz, Poland.
Gawron
Anna
A
Zwolinska
Anna
A
de Graft-Johnson
Jeffrey
J
Krajewski
Wojciech
W
Krol
Maciej
M
Markowski
Jaroslaw
J
Kostka
Tomasz
T
Nowak
Dariusz
D
D000097:Acetonitriles / Q000737:chemistry; D000328:Adult; D000418:Albumins / Q000378:metabolism; D000975:Antioxidants / Q000008:administration & dosage / Q000494:pharmacology*; D001628:Beverages; D001713:Biphenyl Compounds; D011232:Chemical Precipitation; D005260:Female; D005290:Ferric Compounds / Q000378:metabolism; D005502:Food; D016166:Free Radical Scavengers / Q000097:blood* / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D016014:Linear Models; D008297:Male; D027845:Malus; D008875:Middle Aged; D010084:Oxidation-Reduction; D010851:Picrates / Q000097:blood* / Q000378:metabolism* / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013696:Temperature
D000097:Acetonitriles; D000418:Albumins; D000975:Antioxidants; D001713:Biphenyl Compounds; D005290:Ferric Compounds; D016166:Free Radical Scavengers; D010851:Picrates; C004931:1,1-diphenyl-2-picrylhydrazyl; C032159:acetonitrile