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Comparison of antioxidant capacity in urine by ORAC analysis after
consumption of muscadine grape juice B. DeRatt, G. Whitwell, F. X. Arredondo, N. E. Craft North Carolina Wesleyan College and Craft Technologies Inc., Wilson, NC
Approximately 2-3% of oxygen consumed during cellular aerobic
metabolism is converted into free radicals.1 Antioxidants and phenolic
compounds work to remove the free radicals before damage can occur.
Most fruits and vegetables contain high levels of antioxidants.2
Muscadine grapes, which are native to the southeastern United States,
contain high levels of antioxidants. In this study, urine was analyzed
before and after the consumption of muscadine grape juice to compare the
antioxidant statuses. The Oxygen Radical Absorbance Capacity assay
(ORAC) measures the antioxidant scavenging activity against peroxyl
radicals which is induced by 2,2’-Azobis(2-amidinopropane)
dihydrochloride (AAPH).3 ORAC was used to determine the entire
antioxidant status of each sample but uric acid, which is naturally
produced in the body, was found to overshadow the effects of the juice
consumption. The uric acid and vitamin C concentration were determined
for the control and experimental data using high pressure liquid
chromatography.
•As a population, there was no significant change in the antioxidant capacity of
urine before the consumption of grape juice compared to the antioxidant
capacity of urine after grape juice consumption. This was determined by the
ANOVA statistical analysis.
•The experimental t-test analysis showed that two participants had statistical
increases in the antioxidant capacity after consuming the grape juice. This is
shown by the asterisks above the bars in the ORAC graph.
•Uric Acid analysis was performed to determine if the uric acid in the samples
was overshadowing the antioxidant effects from the grape juice. Urinary uric
acid concentration was lower in nearly all subjects after the consumption of
muscadine grape juice. It is unknown if the uric acid decrease is due to juice
consumption but any changes in urinary ORAC due to juice consumption were
obscured by concomitant changes in urinary uric acid.
•Although some people's initial urine sample did not contain Vitamin C, in those
that did, an increase was seen after the consumption of the grape juice.
•The total phenolics analysis of the grape juice indicated that 115.4 µg/g is
available for each sample consumed.
Figure 2. Statistical significance was determined for 2 participants when comparing the averages of the
control and experimental data while 8 participants had no significant increase.
I would like to thank Craft Technologies for their support and the opportunity to
attend this conference. I would like to thank Dr. Craft, Dr. Arredondo, and Dr.
Whitwell for their insight and guidance. I would also like to thank Roger and
Gail Davenport Taylor for their contributions to the Honor’s Program at North
Carolina Wesleyan College.
1 Wu, X. B. (2004). Lipophilic and Hydrophilic Antioxidant Capacities of
Common Foods in the United States. J. Agric. Food Chem. , 4026-4037. 2 Prior, R. H.-W. (2003). Assays for Hydrophilic and Lipophilic Antioxidant
Capacity (oxygen radical absorbance capacity (ORACFL)) of Plasma and Other
Biological and Food Samples. J. Agric. Food Chem. , 3273-3279 3 Ou, B. H.-W. (2001). Development and Validation of an Improved Oxygen
Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent
Probe. J. Agric. Food Chem. , 4619-4626. 4 zenbio. (2008, September 8). ORAC Antioxidant Assay Kit. Retrieved July
2010, from www.zenbio.com
ORAC
•Each urine sample was diluted by 100-fold before performing the ORAC
analysis. Fluorescein was used as the fluorescent probe and Trolox was
the standard. Loss of fluorescence over time is due to the peroxyl-radical
formation by the breakdown of AAPH.4 The results were analyzed in
Excel and statistical analyses was performed with IBM SPSS Statistics
Software.
Uric Acid Analysis
•High Pressure Liquid Chromatography (HPLC) was used to determine
uric acid concentration. Daily aliquots for the control, day 1-10, were
combined so a approximate uric acid content could be determined. The
same protocol was used for experimental days. Vitamin C concentrations
in the grape juice and each sample were also determined in this test.
Total Phenolics
•This spectophotometric test measures all the phenolic compounds in the
sample. The Folin reagent used causes a color change based on the ability
of each sample to be oxidized. A calibration curve of catechin was used to
estimate the phenolic concentration of the muscadine grape juice. The
wavelength used was 765 nm.
INTRODUCTION
METHODS
RESULTS DISCUSSION
BIBLIOGRAPHY
ACKNOWLEDGEMENTS
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Are
a U
nd
er t
he
Cu
rve
Cycle Number
Trolox Calibration
Blank
S1 (200 µM)
S2 (100 µM)
S3 (50 µM)
S4 (25 µM)
S5 (12.5 µM)
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10
OR
AC
(µ
mo
l T
rolo
x/m
L)
Participants
Antioxidant content of urine before and during Muscadine juice
consumption
Avg 1-10
Avg 11-20
*
*
Figure 1. Trolox calibrations were run at the beginning and end of each run.
Figure 3. Uric acid (µg/mL) in combined urine samples by subject and juice consumption status. Generally
higher uric acid content can be seen in the control days. Significance was not determined.
0
100
200
300
400
500
600
700
800
1 2 3 4 5 6 7 8 9 10
ug
/mL
Uri
c A
cid
Particpants
Uric acid content of urine before and during Muscadine juice consumption
Uric Acid Control
Uric Acid with Juice
y = 0.204x - 0.121
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 1 2 3 4 5
Ab
sorb
an
ce
Calibrants
Total Phenolics Determination in
Muscadine Grape Juice
Figure 4. Catechin calibrant curve is used to
determine the micrograms of total phenolic for per
gram of sample.
Figure 5. A chromatogram showing the
Vitamin C and uric acid peaks of one subject.
Uric Acid Analysis Chromatogram
CONCLUSIONS
•Positive variance was found in two participants consistent with a positive
correlation between antioxidant consumption and antioxidant absorption.
• Since uric acid was found to play a major role in total antioxidant capacity,
future studies will account for this contribution.
•The availability of the analytical methods for antioxidant assays in urine and
other biological samples have provided means for observing enhanced antioxidant
concentration in urine.