APPENDIX
BIOMARKERS OF OXIDATIVE STRESS
PARTICIPANTS AND METHODS
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Laboratory measurements of biomarkers of oxidative stress in 42 subjects (19 and 23 in
the ß-glucan and control groups, respectively) were performed at the Antioxidants
Research Laboratory of the Jean Mayer United States Department of Agriculture Human
Nutrition Research Center on Aging at Tufts University (Boston, MA). Oxidized low
density lipoproteins (oxLDL) in serum was measured using an enzyme-linked
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immunosorbent assay (ELISA) kit (ALPCO Diagnostics, Windham, NH) in which two
monoclonal antibodies are directed against separate antigenic determinants on the
oxidized apolipoprotein B molecule. Malondialdehyde (MDA) was assayed in plasma by
treatment with thiobarbituric acid (TBA) and measuring the resulting TBA-MDA adduct
by HPLC equipped with a fluorometric detector (Fukunaga 1998, Hong 2000). Plasma
15
protein carbonyls were assayed by treatment with 2,4-dinitrophenylhydrazine and
measuring the resulting hydrazone spectrophotometrically (Levine 1990). Glycosylated
hemoglobin (HbA1C) was measured in whole blood using a latex enhanced turbidimetric
immunoassay kit (Roche Diagnostics, Indianapolis, IN).
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RESULTS
There were no significant differences in mean serum oxLDL, plasma MDA, plasma
protein carbonyls, or whole blood HbA1c between the oat β-glucan (n = 19) and control (n
= 23) groups overall or within BMI subgroups (see Appendix Table).
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The following observations were made among the 42 subjects at baseline: [i] fasting
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plasma glucose significantly correlated with HbA1c ( = 0.35, P = 0.02) and with MDA (r
= 0.33, P =0.03), but not with other biomarkers of oxidative stress; glucose incremental
AUC correlated with MDA ( = 0.33, P = 0.04), and glucose peak concentration tended
to correlate with MDA (r = 0.30, P = 0.053); [ii] there was a significant correlation
between insulin peak concentration and oxLDL ( = 0.32, P = 0.04), and between insulin
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incremental AUC and oxLDL ( = 0.33, P = 0.03); [iii] there was a trend of a positive
correlation between systolic blood pressure and plasma protein carbonyls (r = 0.29, P =
0.06), and between diastolic blood pressure and plasma protein carbonyls (r = 0.29, P =
0.07) but not other biomarkers of oxidative stress. These data suggest that oxidative
stress is related to carbohydrate metabolism and may be related to blood pressure in these
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subjects.
DISCUSSION
Several classes of compounds with antioxidant activity have been identified in oats
including vitamin E tocols, flavonoids, and non-flavonoid phenolic acids (Collins 1986;
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Collins 1989; Collins 1991; Peterson 1993; Shahidi 1995). Additional investigations
have further characterized the antioxidant capacity of oat fractions, including hulls, bran
and endosperm (Dimberg 1993; Handelman 1999). Avenanthramides and phenolic
acids from oats are bioavailable and act synergistically with vitamin C to enhance
hamster and human LDL resistance to oxidation (Chen 2004). Thus, we hypothesized
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that an increased consumption of oats may reduce oxidative stress in these clinically
obese subjects.
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Obesity is a risk factor for cardiovascular diseases (Kannel 2002; Eckel 2002). The
increased cardiovascular morbidity and mortality in obesity, particularly abdominal
obesity, is partly mediated through a variety of molecular mechanisms linked to platelet
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and vascular abnormalities (Yudkin 2000). Abdominal adiposity is a useful predictor of
vascular endothelial function in healthy overweight adults (Brook 2001) and obesity can
induce endothelial dysfunction via oxidative stress (Perticone 2001). For example, it has
been reported that obese women have higher levels of urinary 8-iso-prostaglandin F2
(8-iso-PGF2, a marker of in vivo lipid peroxidation) and higher levels of 11-dehydro-
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TxB2 (a marker of in vivo platelet activation) than non-obese women (Davi 2002); these
abnormalities are driven by inflammatory triggers related to the degree of abdominal
adiposity and are, at least in part, reversible with a successful weight-loss program. In
the Framingham Study, a community-based cohort of more than 2800 men and women,
obesity was associated with increased urinary 8-iso-PGF2(Keaney 2003). It has been
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reported that surgical weight loss leads to a significant decrease in plasma MDA and a
significant increase in plasma -tocopherol 24 wk after surgery (Kisakol 2002). These
data are consistent with studies in mice and rats that have shown that obesity is associated
with increased oxidative stress (Nakao 2000; Dobrian 2001). In the obese insulinresistant Zucker rat, 8-iso-PGF2 levels were found to be markedly elevated relative to
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controls (Laight 1999).
As no single measure of lipid, protein or DNA oxidation adequately reflects oxidative
stress, we elected to use measures of lipid peroxidation (i.e., oxLDL and MDA) and
protein oxidation (i.e., protein carbonyls) as relevant to this study. The detection of
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circulating autoantibodies against oxLDL reflects the occurrence of chronic in vivo LDL
oxidation processes. Elevated levels of autoantibodies against oxLDL have been detected
in patients with coronary artery disease (Parums 1990) and in type 2 diabetics (Hsu
2002). Protein carbonyls are sensitive markers of oxidative injury and oxidant stress and
are formed through the oxidation of proteins by a variety of mechanisms. While HbA1C
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is a marker of glycemic control, it is also a marker of the production of a heterogeneous
group of irreversible complex protein adducts, i.e., advanced glycation end products
(AGE), which generate reactive oxygen species (Makita 1992).
The null differences in biomarkers of oxidative stress between the oat β-glucan and
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control groups in this study could be due to a number of factors including the small
sample size, as the power calculations for the protocol were based on the primary
outcome of blood pressure, and not on oxidative stress measures. The null outcome
could also be due to an inadequate dose of oat antioxidants, as the previous in vitro and
animal model studies demonstrating a reduction in biomarkers of oxidation were
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conducted with extracts of oat bran (Chen 2004), and thus, with a higher concentration of
phenolics and tocols than found in the oat products provided to the subjects in the present
study. Furthermore, an insufficiently elevated level of oxidative stress may have been
common among the subjects, as most antioxidant interventions are only successful in
lowering oxidative stress when it is elevated.
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