European Journal of Clinical Nutrition (2015) 69, 1256–1261

© 2015 Macmillan Publishers Limited All rights reserved 0954-3007/15

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ORIGINAL ARTICLE
Association of oxidative stress biomarkers with adiposity and
clinical staging in women with breast cancer
AAF Carioca1, SMML Verde1,2, LA Luzia1, PHC Rondó1, MRDO Latorre3, THP Ellery1 and NRT Damasceno1

BACKGROUND/OBJECTIVES: Breast cancer is a disease characterised by both oxidative reactions and inflammation. However, few
studies have focused on the oxidative and inflammatory biomarkers. The aim of the present study was to evaluate the association
between oxidative stress markers and adiposity and clinical staging, as well as the association between the oxidative and the
antioxidant biomarkers of women with breast cancer.
SUBJECTS/METHODS: A total of 135 cases of breast cancer occurring in 2011 and 2012 were assessed. After exclusions, 101 pre-
and post-menopausal women with clinical staging I to IV were eligible to participate in the study. The anthropometric evaluation
was performed by collecting data on waist circumference, body mass index and body composition. The socioeconomic and clinical
profiles were determined using a standard questionnaire. For the oxidative biomarkers, thiobarbituric acid reactive substances
(TBARS), oxidative DNA damage (8-hydroxy-2-deoxyguanosine (8-OHdG)), low-density lipoprotein(− ) (LDL( − )), autoantibody
anti-LDL( − ) and liposoluble antioxidants (α-tocopherol, retinol and β-carotene) were analysed. The data were analysed using
differences in the mean values, correlation tests and multiple linear regression.
RESULTS: The antioxidant levels were higher in postmenopausal women with clinical staging I and II and negative lymph nodes.
The TBARS level was associated with clinical staging. Adiposity was associated with levels of retinol and 8-OHdG, whereas LDL( − ),
8-OHdG and TBARS were correlated with liposoluble antioxidants after adjusting for the confounders.
CONCLUSIONS: The adiposity and clinical staging of patients were associated with oxidative stress. The oxidative and antioxidant
biomarkers showed a negative correlation in patients with breast cancer.
European Journal of Clinical Nutrition (2015) 69, 1256–1261; doi:10.1038/ejcn.2015.84; published online 3 June 2015

INTRODUCTION dietary profile, the antioxidant capacity of nutrients and other

Breast cancer is one the most prevalent types of cancer, with high
incidence and mortality among women worldwide.1 An estimated
235 030 new cases and 40 430 deaths from breast cancer have
been reported in the USA.2 Brazil reflects a similar pattern, and in
2014, the expected incidence of breast cancer was 56.09 new
cases per 100 000 women.3
Despite the high prevalence of the disease, the prevention,
early detection and adequate treatment have helped to delay
tumour recurrence and have contributed to improving the
patients’ quality-of-life. These benefits are directly dependent on
the knowledge of the mechanism involved in the pathogenesis of
breast cancer. In this condition, the redox homeostasis is disrupted
and is related to an increase in oxidative stress.4 The continuous
overproduction of free radicals and reactive metabolites along
with the decrease of their elimination, can allow the activation of
the transcription factors that regulate several genes involved in
chronic inflammation, thus contributing to several steps of
carcinogenesis.5,6 The imbalance between oxidative species and
antioxidant system induced by exogenous (diet and smoking) or
endogenous (oestrogen) factors promotes oxidative stress during
the initiation, promotion and progression of breast cancer.6–8
According to the American Institute for Cancer Research (AICR),1
observational studies have shown that a dietary pattern rich in
vegetables, fruit, dairy products, fish, poultry and low fat is
associated with a lower risk of breast cancer. Regarding this
bioactive components represent a potential protective mechan-
ism against the development of cancer.7–9
Therefore, the evaluation of antioxidants, oxidative biomarkers
and inflammatory status is crucial in clinical practice. 8-hydroxy-2-
deoxyguanosine (8-OHdG), malondialdehyde and antioxidant
vitamins have played an important role in assessing the prognosis
and the risk for the initiation and recurrence of breast cancer.9–11
More recently, Hursting and Dunlap12 proposed that the
relationship between cancer and oxidation can change in obesity,
probably because of the mechanism involved in the development
of obesity, in which a chronic and low intensity inflammatory
process occurs. Obese women may likely have a metabolic
perturbation for oxidative reactions and for the development of
breast cancer. Despite this hypothesis, few studies have investi-
gated the impact of adiposity and clinical profile on the
relationship with oxidative stress.13–17 Therefore, the aim of the
present study was to evaluate the association of oxidative stress
markers and adiposity and clinical staging, as well as the
association between the oxidant and the antioxidant biomarkers
in Brazilian women with breast cancer.
MATERIALS AND METHODS
Subjects
The women were recruited from the General Hospital of Fortaleza (Ceará,
Brazil), after their medical appointment at the mastology clinic for

1
Department of Nutrition, School of Public Health, University of São Paulo, São Paulo, Brazil; 2Nutrition course, University of Fortaleza, Fortaleza, Brazil and 3Department of
Epidemiology, School of Public Health, University of São Paulo, São Paulo, Brazil. Correspondence: Professor NRT Damasceno, Department of Nutrition, School of Public Health,
University of Sao Paulo. Avenida Dr Arnaldo, 715, Sao Paulo 01246-904, Brazil.
E-mail: nagila@usp.br
Received 18 December 2014; revised 11 April 2015; accepted 19 April 2015; published online 3 June 2015

preventative care of breast cancer in 2011 and 2012. The women with
recent clinical and histopathological diagnosis of breast cancer, clinical
staging I to IV, without previous cancer treatment or metastasis, and with a
Karnofsky Performance Scale Index score of 470 (patients who are able to
conduct normal activity and to work and who require no special care)18
were selected for inclusion in the study. The exclusion criteria include
metastatic cancer and use of vitamin supplements. This study was
approved by the Research Ethics Committees of the General Hospital of
Fortaleza (No. 050507/10) and the School of Public Health, the University of
São Paulo (No. COEP 2162), and written informed consent was obtained
from all of the patients. The data were collected according to good clinical
practice. Before commencing the study, a training workshop was held to
establish standard operating procedures among all of the researchers
involved.
Socioeconomic and clinical profiles
The socioeconomic profile (age, marital status, race, education and family
income) of the patients was determined by a direct interview assessment
using a standard questionnaire. The patient’s clinical history was obtained
from the medical records and was conducted by a direct face-to-face
interview. The following items were assessed: menopausal status
(menopause was defined as 12 months of amenorrhea followed by
reduced ovarian hormonal activity in the absence of surgical or hormonal
induction),19 nulliparity, hormone replacement therapy, breastfeeding
( ⩾3 months), smoking (cigarette, cigar or pipe smoking one or more
times during the 30 days leading up to the data collection),20 alcohol
intake (415 g of ethanol/day,21 tumour subtype, lymph nodes and
tumour size.
Anthropometry and adiposity
Measurements of weight, height, waist circumference (WC) and body
composition were obtained from the patients. The patient’s weight was
determined using a digital scale (Control, Plenna, São Paulo, Brazil), with
150 kg capacity and 100 g precision. The patient’s height was measured
using a stadiometer (Alturexata, Minas Gerais, Brazil) with a limit of 2.10 m
and precision of 1 mm. Weight and height were utilised to calculate the
body mass index (kg/m2), and the patients were classified according to
WHO (World Health Organization) recommendations22 and the study by
Lipschitz.23 WC was measured at the level of the umbilicus, using an
inelastic measuring tape, and the cardiometabolic risk of the patients was
calculated according to the WHO classification.22 The body composition
was evaluated using bioelectrical impedance for estimating the percen-
tage of fat mass (tetrapolar bioimpedance Biodynamic Model 450 device,
São Paulo, Brazil). All of the measurements were collected in duplicate by a
trained interviewer. The fat mass percentage (%FM) was classified
according to the study by Lohman.24
Blood collection
After a 12-h fast, the blood samples were obtained by peripheral venous
puncture and were collected in vacutainer tubes containing EDTA (1 mg/ml)
used as an anticoagulant and antioxidant; the blood samples were stored
in ice and protected from light until obtention of plasma (1500 g, 10 min,
4 °C). The following protease inhibitors were added to the plasma:
aprotinin (2 μg/ml), benzamidine (2 mM) and phenylmethanesulfonyl
fluoride (1 mM) and the antioxidant butylated hydroxytoluene (20 mM).
The plasma was aliquoted and stored at − 80 °C until analysis.
Thiobarbituric acid reactive substances
Plasma (50 μl) was added to 1 ml of thiobarbituric acid (0.046 M),
trichloroacetic acid (0.92 M) and hydrochloric acid (0.25 M) and subse-
quently incubated in a boiling water bath (100 °C) for 30 min. After this
period, samples were centrifuged at 4 °C for 15 min at 8000 g, and
supernatant (200 μl) was determined by ultraviolet absorption at 535 nm.
Quantification was performed using the standard curve with 1,1,3,3
tetraethoxypropane (0.2–4.0 μmol of thiobarbituric acid reactive sub-
stances (TBARS)/L).25 The results were expressed as micromole TBARS
per milligram of protein. All of the analyses were performed in duplicate.
The intra- and inter-coefficients of variation were 2.8% and 7.2%,
respectively.
© 2015 Macmillan Publishers Limited
Oxidative stress biomarkers in breast cancer
AAF Carioca et al
1257
Oxidative DNA damage and inflammation biomarkers
The analysis of 8-OHdG was performed using a competitive ELISA kit
(EIA DNA Damage, Enzo Life Sciences, Inc., Farmingdale, NY, USA) and
adiponectin was analysed using sandwich enzyme immunoassay by
Human Adiponectin ELISA (Enzyme-Linked Immunosorbent Assay) kit
(EMD Millipore, St Charles, MO, USA).
LDL(− ) and anti-LDL( − ) autoantibody detection
Low-density lipoprotein( − ) (LDL( − )) was detected using sandwich
enzyme immunoassay ELISA, according to a standardised protocol by
Faulin et al.26 The results were expressed as the mean absorbance minus
the background sample and were subsequently applied to the standard
curve and multiplied by the corresponding dilution. A calibration curve
was conducted for each plate, using LDL( − ) extracted from the human
plasma by fast protein liquid chromatography as a standard (0.6–20 μg/ml).
The results were expressed as unit per litre with litre representing 1.0 g/l
oxidised Apo B. The intra- and inter-coefficients of variation were 4.8% and
8.9%, respectively.
The anti-LDL( − ) autoantibody was detected using the ELISA capture
antibody. The LDL( − ) isolated using fast protein liquid chromatography
and ELISA method were performed according to the method previously
described.27 The results were determined by applying the average
absorbance of sample background to the equation of the standard curve
monoclonal antibody (0.004–0.125 mU/l). The intra- and inter-coefficients
of variation were 2.8 and 6.6%, respectively.
Antioxidant analysis
The liposoluble antioxidants (α-tocopherol, retinol and β-carotene) were
extracted from plasma (200 μl) by the addition of 200 μl ethanol solution
(Merck, Darmstadt, Germany; high-performance liquid chromatography
(HPLC)) followed by 5 s of vortex (Biomixer-MVS-1, Hamilton Beach,
Washington, NC, USA). After this period, 500 μl of hexane (Merck; HPLC)
was added and the final solution was mixed (2 min). The solution was
centrifuged (Centrifuge Eppendorf 5415C, Eppendorf AG, Hamburg,
Germany) for 5 min at 700 g at 4 °C, and supernatant (250 μl) was
transferred and evaporated under nitrogen. The extracted antioxidants
were resuspended in 200 μl of mobile phase consisting of acetonitrile/
methanol/dichloromethane (70%:20%:10%).
The analysis of α-tocopherol, retinol and β-carotene was performed
using a HPLC (Shimadzu Inc., Tokyo, Japan) that was conducted through a
separation column Synergy Fusion 5μ—C8(ref. 2) 150 ×4.6 mm column and
Security Guard-HPLC Guard Cartridge System-KJO 4282 (pre-column) both
from Phenomenex (Torrance, CA, USA). The effluent was monitored by
SPD-10AVVP UV–vis and RF-10AXL (Shimadzu Inc.). The fluorescence
detector was set at 295 nm (excitation) and 340 nm (emission). The flow of
the mobile phase was 1.0 ml/min for 8 min. Retention times for
α-tocopherol, retinol and β-carotene were 4.5 min, 2.5 min and 6.7 min,
respectively. The curve was constructed using external standard (99%)
with concentrations of α-tocopherol between 2 and 49 μmol/l, retinol
between 0.2 and 6.5 μmol/l and β-carotene between 0.1 and 0.76 μmol/l.
The coefficient of variation was 2.9%.
Statistical analysis
The results were evaluated using the Statistical Package for the Social
Sciences (SPSS Inc., Chicago, IL, USA) version 20.0. Adherence to the
normal curve was evaluated using the Kolmogorov–Smirnov test (P40.05).
Pearson’s correlation coefficients were calculated. The comparison of
biochemical parameters between the groups was performed using
Student’s t-test. The multiple linear regression models were used to
evaluate the associations between dependent variables (LDL(− ), anti-LDL
( − ) autoantibody, TBARS and 8-OHdG), whereas the antioxidants were
considered to be independent variables. These variables were tested using
a crude model (model 1) and models controlled for potential confounders:
model 2—clinical staging (I and II or III and IV) and model 3—body fat
percentage ( o32 or ⩾32%). Values of Po0.05 were considered to be
statistically significant.
RESULTS
A total of 135 women with a mean age of 50.4 (11.3) years were
initially contacted to participate in the study; however, 13 women
proved to be ineligible. The women were excluded during follow-up
European Journal of Clinical Nutrition (2015) 1256 – 1261
 Oxidative stress biomarkers in breast cancer
AAF Carioca et al
1258
for the following reasons: hospital transfers (n = 8), refusals (n = 5) The relationship between menopausal status and oxidative
and pretreatment of cancer or benign breast disease (n = 8). stress was previously described in the study by Victorino et al.17
Therefore, the final sample consisted of 101 patients. The majority According to the authors, the post-menopausal women had a
of the participants were premenopausal (50.5%) and 65.4% had more favourable antioxidant profile, contributing to lower levels of
clinical staging I and II (Table 1). The patients had a mean body oxidative damage and higher antioxidant capacity. Although the
mass index of 27.9 (4.6) kg/m2, mean %FM of 35.3 (4.8)% and mechanism involved remains unclear, Okoh et al.28 proposed that
mean WC of 96.1 (10.7) cm. The adiposity was not associated with oestrogen stimulates the oxidation, contributing to genomic
clinical staging in this study (χ2 = 1.720; P = 0.190). instability. This observation was confirmed by Fusell et al.,29 who
The values of LDL(− ), autoantibody anti-LDL( − ) and 8-OHdG demonstrated that the production of reactive oxygen species in
showed no significant differences according to the clinical profile. the mammary epithelium is elevated. In addition, these women
However, the TBARS levels were higher in the advanced clinical exhibited higher levels of plasma α-tocopherol and retinol.
stage (P = 0.037). Furthermore, the mean values of α-tocopherol Regarding the negative role of oxidative stress in the
(P = 0.002) and retinol levels (P o0.001) were higher among the development of cancer, previous studies have shown that obesity
postmenopausal women. The β-carotene level showed differences in post-menopausal women can accelerate the reactive oxygen
species generation.14,30,31 Our results confirmed that higher values
as a function of clinical staging (P = 0.014), whereas the retinol
of 8-OHdG were observed in women with a large WC, whereas
levels differed with lymph node involvement (P = 0.017) (Table 2).
lower levels of retinol were correlated with an overweight status.
Women with a large WC had higher 8-OHdG concentrations
8-OHdG has been widely used as a biomarker of oxidative stress,
(P = 0.020), whereas those with excess weight had a lower
and breast cancer has been considered to be a prognostic factor
concentration of retinol (P = 0.019). Adiponectin concentrations because a more aggressive tumour profile was associated with
were higher in patients without excess weight (P = 0.002) (Table 3). 8-OHdG serum levels and expression.10
LDL( − ) was inversely correlated with α-tocopherol (r = −0.28, In addition to the menopausal status, clinical staging also exerts
P = 0.005) and retinol (r = − 0.24, P = 0.017). The autoantibodies a significant impact on oxidative stress. Kumaraguruparan et al.13
showed no significant correlation. 8-OHdG was inversely corre- observed that the oxidant/antioxidant ratio changes as a function
lated with β-carotene (r = − 0.45, P o0.001) (Table 4). of normal or tumour tissue and clinical staging. Our results
The relationship of α-tocopherol with LDL(− ) remained even showed that the advanced products of lipid peroxidation (TBARS)
after adjusting for confounders. The association of retinol with LDL were higher in women with clinical staging III and IV and that the
( − ) was independent of %FM and clinical staging. None of the β-carotene levels had reduced. Recently, Panis et al.16 verified that
antioxidants were associated with the anti-LDL( − ) autoantibody. at advanced clinical stages, pro-inflammatory and oxidative
The β-carotene level was associated with 8-OHdG and TBARS after stimuli are present. Further supporting the oxidative basis of
adjusting for %FM and clinical staging (Table 5). cancer, Pande et al.32 concluded that women with advanced
tumour stages and positive lymph nodes had lower levels of
antioxidant vitamins, a finding that was corroborated by the
DISCUSSION results described in the present study. According to Pande et al.,32
Our results confirmed that antioxidants are negatively correlated the imbalances observed in the antioxidants and oxidants are
with oxidative biomarkers and that oxidative stress is associated related to the prognosis and clinical tumour stage. This association
with menopausal status, clinical staging and adiposity of women is directly dependent on the activation of the NF-kB p65 subunit,
with breast cancer. causing an imbalance in the homeostasis of the intracellular redox
environment.32
In the present study, β-carotene was the main antioxidant
Table 1. Socioeconomic and clinical characteristics of patients with
related to the oxidative biomarkers. This profile has been
breast cancer (n = 101 women)
previously described;7,9,33 however, to the best of our knowledge,
Variables n % this is the first time that the antioxidant has been simultaneously
associated with oxidative DNA damage (8-OHdG) and lipid
Race peroxidation (TBARS) in women with breast cancer. In addition,
White 26 25.7 we have shown that LDL( − ) and α-tocopherol and retinol are
Non-white 75 74.3 inversely correlated. To date, these associations have not been
reported in the literature although a relationship between LDL( − )
Per capita income and α-tocopherol was previously observed in obese adolescents,34
o1 BMW 61 60.4
⩾1 BMW 40 39.6 patients submitted to haemodialysis35 and subjects with
Postmenopausal (yes) 50 49.5 dyslipidaemia.36 Delimares et al.37 observed that the oxLDL
Nulliparity (yes) 19 18.6 generation was associated with an increased risk of breast cancer
Hormone replacement therapy (yes) 8 7.9 (odds ratio = 1.02, 95% confidence interval = 1.001–1.031), sug-
Breastfeeding (yes) 68 67.3 gesting that elevated oxLDL levels, indicating the involvement of
Smoking (yes)a 9 8.9 lipid peroxidation, can lead to higher oxidative stress. Recently,
Alcohol intake (yes)b 18 17.8 Khaidakov et al.38 demonstrated pathways involved in breast
Family history of breast cancer (yes) 70 69.3 cancer through the uptake of oxidised-LDL, with the stimulation of
Subtype of breast cancer (ductal) 74 73.3 miR-21, an oncogene linked to carcinogenesis.
Lymph nodes (yes) 26 25.7
Clinical stage (I and II) 66 65.4
The LDL particles can be modified, resulting in a more
BMI (overweight)c 68 67.3 electronegative LDL subfraction with oxidised characteristics39
WC (⩾88 cm) 75 74.3 and proinflammatory properties.40 Previously, we proposed that
%FM (⩾32%) 77 76.2 LDL( − ) is a potential early biomarker for oxidative stress;34 this
likelihood could be reinforced by their antibodies anti-LDL( − ). The
Abbreviations: BMI, body mass index; BMW, brazilian minimum wage
present study confirms that women with breast cancer are in
(equivalent to 311 USD per month); WC, waist circumference; %FM, fat
mass percentage. aSmoked a cigarette, cigar or pipe on ⩾ 1 day during the
oxidative stress and show modified particles with different levels
30 days before data collection. bAlcohol intake considered ⩾ 15 g of of oxidative damage.
ethanol/day. cOverweight ⩾ 25 kg/m2 (adults)22 and ⩾ 27 kg/m2 (elderly).23 The patient’s clinical profile and adiposity influenced the
correlations between the biomarkers, where a significant

European Journal of Clinical Nutrition (2015) 1256 – 1261 © 2015 Macmillan Publishers Limited
 Oxidative stress biomarkers in breast cancer
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Table 2. Oxidative and antioxidant parameters in patients with breast cancer according to their clinical profiles

Total Menopausal status Clinical staging Lymph node

Premenopausal (n = 51) Postmenopausal (n = 50) I/II (n = 66) III/IV (n = 35) Yes (n = 26) No (n = 75)

Oxidative parameters
LDL(− ), U/la 4.2 (5.4) 4.3 (5.8) 4.1 (5.1) 4.1 (4.7) 3.9 (4.0) 3.9 (4.3) 4.6 (5.4)
Autoantibody anti-LDL( − ), mU/la 4.6 (3.0) 5.0 (3.3) 4.1 (2.6) 4.7 (3.5) 4.1 (2.0) 4.3 (2.3) 3.7 (1.8)
8-OHdG, ng/ml 18.2 (6.0) 17.2 (6.0) 19.3 (5.7) 17.7 (5.4) 18.3 (6.0) 17.3 (5.6) 19.0 (5.4)
TBARS, μmol per mg of protein 0.8 (0.2) 0.8 (0.2) 0.8 (0.2) 0.8 (0.2) 0.9 (0.2)d 0.8 (0.2) 0.8 (0.2)

Antioxidant parameters
α-tocopherol, μmol/l 11.3 (2.8) 10.4 (2.1) 12.1 (3.1)b 11.4 (2.7) 10.6 (2.7) 11.6 (2.8) 10.7 (2.7)
β-carotene, μmol/la 0.5 (0.4) 0.4 (0.3) 0.5 (0.4) 0.5 (0.4) 0.3 (0.2)e 0.5 (0.4) 0.4 (0.3)
Retinol, μmol/l 1.7 (0.5) 1.5 (0.4) 1.8 (0.5)c 1.7 (0.5) 1.5 (0.3) 1.7 (0.5) 1.5 (0.4)f

Adipocytokine
Adiponectin, μg/mla 8.8 (5.3) 9.3 (5.6) 8.3 (4.9) 9.1 (5.6) 8.5 (5.5) 9.4 (5.8) 9.0 (5.7)
a
Abbreviations: LDL, low-density lipoprotein; TBARS, thiobarbituric acid reactive substance; 8-OHdG, 8-hydroxy-2-deoxyguanosine. Variable logarithm.
b
P ¼ 0.002. cPo0.001. dP ¼ 0.037. eP ¼ 0.014. fP ¼ 0.017. Po 0.05 was considered to be statistically significant. Student’s t-test was used for the variable and the
results are expressed as the mean and s.d. of values. Values in bold denote significant differences.

Table 3. Oxidative and antioxidant parameters in patients with breast cancer according to adiposity level

BMIa WC %FM

Without excess weight With excess weight o88 cm ⩾ 88 cm o32% ⩾ 32%

(n = 33) (n = 68) (n = 20) (n = 75) (n = 24) (n = 77)

Oxidative parameters
LDL( − ), U/lb 5.3 (8.2) 3.7 (3.3) 6.2 (7.3) 3.4 (2.9) 6.0 (7.8) 3.7 (4.3)
Autoantibody anti-LDL( − ), 4.4 (2.1) 4.6 (3.4) 4.6 (2.0) 4.7 (3.3) 4.3 (2.2) 4.7 (3.2)
mU/lb
8-OHdG, ng/ml 17.2 (6.5) 18.8 (5.6) 16.0 (4.7) 19.3 (6.1)e 16.5 (5.5) 18.7 (6.0)
TBARS, μmol per mg of 0.8 (0.3) 0.8 (0.2) 0.8 (0.3) 0.8 (0.2) 0.8 (0.2) 0.8 (0.2)
protein

Antioxidant parameters
α-tocopherol, μmol/l 11.7 (3.1) 11.0 (2.6) 10.5 (2.3) 11.5 (2.9) 10.8 (2.0) 11.4 (3.0)
β-carotene, μmol/lb 0.6 (0.4) 0.4 (0.3) 0.5 (0.4) 0.4 (0.3) 0.5 (0.3) 0.5 (0.4)
Retinol, μmol/l 1.8 (0.4) 1.6 (0.5)c 1.7 (0.4) 1.6 (0.5) 1.5 (0.4) 1.7 (0.5)

Adipocytokine
Adiponectin, μg/mlb 11.3 (6.7) 7.7 (4.0)d 10.1 (5.9) 8.2 (4.6) 9.9 (6.0) 8.5 (5.0)
Abbreviations: BMI, body mass index; LDL, low-density lipoprotein; WC, waist circumference; TBARS, thiobarbituric acid reactive substance; %FM, fat mass
percentage; 8-OHdG, 8-hydroxy-2-deoxyguanosine. aOverweight ⩾25 kg/m2 (adults)22 and ⩾ 27 kg/m2 (elderly).23 bVariable logarithm. cP ¼ 0.019. dP ¼ 0.002.
e
P ¼ 0.020. Po 0.05 was considered to be statistically significant. Student’s t-test was used for the variable and the results are expressed as the mean and s.d.
values. Values in bold denote significant differences.

Table 4. Correlation between the oxidative and the antioxidant parameters

Antioxidant parameters Oxidative parameters

LDL(− ), U/la Autoantibody anti-LDL( − ), mU/la 8-OHdG, ng/ml TBARS, μmol per mg of protein
r (p) r (p) r (p) r (p)

α-tocopherol, μmol/l −0.28 (0.005) − 0.11 (0.264) 0.05 (0.669) − 0.08 (0.409)
Retinol, μmol/l − 0.24 (0.017) 0.01 (0.935) 0.01 (0.916) − 0.04 (0.691)
β-carotene, μmol/la 0.04 (0.734) 0.03 (0.794) −0.45 ( o0.001) −0.27 (0.007)
Abbreviations: LDL, low-density lipoprotein; TBARS, thiobarbituric acid reactive substance; 8-OHdG, 8-hydroxy-2-deoxyguanosine. aVariable logarithm.
Pearson’s correlation was employed for all of the variables. Results were considered significant when P o0.05. Values in bold denote significant correlation.

© 2015 Macmillan Publishers Limited European Journal of Clinical Nutrition (2015) 1256 – 1261
 Oxidative stress biomarkers in breast cancer
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Table 5. Linear regression models for the oxidative and antioxidant variables

Models Crude Model 1 Model 2

Beta 95% CI Beta 95% CI Beta 95% CI

a
LDL(− ) , U/l
α-tocopherol, μmol/l − 0.05 ( − 0.07, − 0.02) − 0.04 ( − 0.08, − 0.01) − 0.04 ( − 0.07, − 0.02)
Retinol, μmol/l − 0.21 ( − 0.38, − 0.04) − 0.18 ( − 0.36, − 0.01) − 0.20 ( − 0.37, − 0.02)
β-carotenea, μmol/l 0.08 ( − 0.16,0.32) 0.00 ( − 0.27,0.27) 0.05 ( − 0.18,0.30)

Autoantibody Anti-LDL( − )a, mU/l

α-tocopherol, μmol/l -0.01 ( − 0.03,0.01) − 0.02 ( − 0.04,0.01) − 0.01 ( − 0.03,0.01)
Retinol, μmol/l 0.00 ( − 0.18,0.12) − 0.02 ( − 0.13,0.10) − 0.01 ( − 0.11,0.11)
β-carotene , μmol/l
a
0.05 ( − 0.10,0.20) 0.07 ( − 0.10,0.25) 0.05 ( − 0.10,0.20)

8-OHdG, ng/ml
α-tocopherol, μmol/l 0.10 ( − 0.37,0.57) 0.09 ( − 0.41,0.59) 0.08 ( − 0.39,0.55)
Retinol, μmol/l 0.16 ( − 0.91,3.24) − 0.01 ( − 2.96,2.95) − 0.09 ( − 3.17,2.98)
β-carotenea, μmol/l − 7.25 ( − 10.62,-3.88) − 7.54 ( − 11.17, − 3.91) − 7.04 ( − 10.42, − 3.66)

TBARS, μmol per mg of protein

α-tocopherol, μmol/l − 0.01 ( − 0.02,0.01) − 0.01 ( − 0.03,0.01) − 0.01 ( − 0.02,0.01)
Retinol, μmol/l − 0.02 ( − 0.11,0.07) − 0.02 ( − 0.10,0.07) − 0.02 ( − 0.11,0.07)
β-carotenea, μmol/l − 0.19 ( − 0.30, − 0.07) − 0.18 ( − 0.30, − 0.05) − 0.19 ( − 0.31, − 0.07)
Abbreviations: CI, confidence interval; LDL, low-density lipoprotein; TBARS, thiobarbituric acid reactive substance; 8-OHdG, 8-hydroxy-2-deoxyguanosine.
a
Variable logarithm. Model 1, adjusted for clinical staging (I and II or III and IV); Model 2, body fat percentage ( o32% or ⩾32%).

association was observed in LDL( − ) with α-tocopherol and retinol
and the percentage of fat. This observation may likely be related
to the fact that LDL( − ) is a biomarker of the initial oxidation
process,41 unlike TBARS and 8-OHdG.
Previous studies in the literature have shown that oxidative
stress in cancer is related to negative tumoural characteristics and
clinical prognosis.13–17 Regarding this background, our results
confirm the importance of antioxidants, such as potential
quencher of free radicals. To translate this scenario into clinical
practice, it is likely that women with breast cancer may benefit
from the increased intake of dietary antioxidants.
We also evaluated adiponectin, an anti-inflammatory biomarker
and an adipocytokine related to adipose tissue and breast
cancer.42 The circulating levels of this adipokine are negatively
correlated with adiposity.12 In this study, women with higher body
mass index had low plasmatic adiponectin concentrations as
expected.
A limitation of this study lies in its design, which precludes the
establishment of a cause–effect relationship. However, an
important strength of this study is that antioxidants were
evaluated using a direct method, thereby avoiding systematic
and randomised bias, a common occurrence in evaluations of
diet.43,44 These limitations are particularly relevant in studies
where the number of subjects included is small and statistical
analysis is not able to reduce these biases. The data of plasma
antioxidants are good tools for measuring the status of these
nutrients at the tissue level; however, we should be cautious in
determining these results as biomarkers of dietary intake because
plasma levels are not reflective of the intake, due to a series of
bioavailability processes that involve such nutrients.45,46 Several
dietary surveys, such as the food frequency questionnaire, have
already been validated using such plasma nutrients,47,48 and good
correlations with a plasmatic concentration have been observed
with the consumption of fruits and vegetables.45,49
Finally, in women with breast cancer, the impact of antioxidants
goes beyond the association with the oxidative biomarkers.
Regression models broaden this view, showing that non-
modifiable (clinical staging) and modifiable (%FM) factors do not
change the association between the antioxidant and the oxidative
products. Therefore, we conclude that women with breast cancer
are subject to oxidative stress and that this profile is directly
influenced by clinical characteristics and adiposity.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This study was supported, in part, by the Sao Paulo Research Foundation (FAPESP),
Brazil (grant 2010/19207-6; 2011/15590-2) and also, in part, by FAPESP process:
2010/19207-6; 2011/15590-2.
AUTHOR CONTRIBUTIONS
AAFC performed data analysis and drafted the manuscript. SMMLV was
involved in data collection and analysis. LAL, MdRDdOL, PHdCR and THPE were
involved in data analysis and critical analysis. NRTD performed data analysis and
final revision and critical analysis of the manuscript. All authors read and
approved the final draft of the manuscript.
REFERENCES
1 American Institute for Cancer Research (AICR). World Cancer Research Fund. Food,
Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective. AICR:
Washington, DC, USA, 2007.
2 Siegel R, Jiemin M, Zhaohui Z, Ahmedin J. Cancer statistics2014. CA Cancer J. Clin.
64: 9–29.
3 Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA). Estimativa 2014:
Incidência de Câncer no Brasil. INCA, Rio de Janeiro, Brazil, 2014. Available at
http://www.inca.gov.br/estimativa/2014/.
4 Ramírez-Expósito MJ, Sánchez-López E, Cueto-Ureña C, Dueñas B, Carrera-Gon-
zález P, Navarro-Cecilia J et al. Circulating oxidative stress parameters in pre-
and post-menopausal healthy women and in women suffering from breast
cancer treated or not with neoadjuvant chemotherapy. Exp Gerontol 2014; 58C:
34–42.
5 Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress,
inflammation, and cancer: how are they linked? Free Radic Biol Med 2010; 49:
1603–1616.
6 Vera-Ramirez L, Sanchez-Rovira P, Ramirez-Tortosa MC, Ramirez-Tortosa CL,
Granados-Principal S, Lorente JÁ et al. Free radicals in breast carcinogenesis,

European Journal of Clinical Nutrition (2015) 1256 – 1261 © 2015 Macmillan Publishers Limited

breast cancer progression and cancer stem cells. Biological bases to develop
oxidative-based therapies. Crit Rev Oncol Hematol 2011; 80: 347–368.
7 Yeon J-Y, Suh Y-J, Kim S-W, Baik H-W, Sung C-J, Kim H-S et al. Evaluation of dietary
factors in relation to the biomarkers of oxidative stress and inflammation in breast
cancer risk. Nutrition 2011; 27: 912–918.
8 Jerby L, Wolf L, Denkert C, Stein GY, Hilvo M, Oresic M et al. Metabolic associations
of reduced proliferation and oxidative stress in advanced breast cancer. Cancer
Res 2012; 72: 5712–5720.
9 Thomson CA, Stendell-Hollis NR, Rock CL, Cussler EC, Flatt SW, Pierce JP. Plasma
and dietary carotenoids are associated with reduced oxidative stress in women
previously treated for breast cancer. Cancer Epidemiol Biomarkers Prev 2007; 16:
2008–2015.
10 Sova H, Jukkola-Vuorinen A, Puistola U, Kauppila S, Karihtala P. 8-Hydro-
xydeoxyguanosine: a new potential independent prognostic factor in
breast cancer. Br J Cancer 2010; 102: 1018–1023.
11 Pande D, Negi R, Karki K, Khanna S, Khanna RS, Khanna H. Oxidative damage
markers as possible discriminatory biomarkers in breast carcinoma. Transl Res
2012; 160: 411–418.
12 Hursting SD, Dunlap SM. Obesity, metabolic dysregulation, and cancer: a growing
concern and an inflammatory (and microenvironmental) issue. Ann NY Acad Sci
2012; 1271: 82–87.
13 Kumaraguruparan R, Kabalimoorthy J, Nagini S. Correlation of tissue lipid perox-
idation and antioxidants with clinical stage and menopausal status in patients
with adenocarcinoma of the breast. Clin Biochem 2005; 38: 154–158.
14 Pansini F, Cervellati C, Guariento A, Stacchini MA, Castaldini C, Bernardi A et al.
Oxidative stress, body fat composition, and endocrine status in pre-and
postmenopausal women. Menopause 2008; 15: 112–118.
15 Badid N, Ahmed FZB, Merzouk H, Belbraouet S, Mokhtari N, Merzouk SA et al.
Oxidant/antioxidant status, lipids and hormonal profile in overweight women
with breast cancer. Pathol Oncol Res 2010; 16: 159–167.
16 Panis C, Victorino V, Herrera A, Freitas L, De Rossi T, Campos F et al. Differential
oxidative status and immune characterization of the early and advanced stages of
human breast cancer. Breast Cancer Res Treat 2012; 133: 881–888.
17 Victorino V, Panis C, Campos F, Cayres R, Colado-Simão A, Oliveira S et al.
Decreased oxidant profile and increased antioxidant capacity in naturally
postmenopausal women. Age 2012; 35: 1–11.
18 Karnofsky DA, Burchenal JHThe clinical evaluation of chemotherapeutic
agents in cancerIn:MacLeod CM(ed) Evaluation of Chemotherapeutic Agents.
Columbia University Press: New York, NY, USA, 1949 pp 191–205.
19 Soules MR, Sherman S, Parrott E, Rebar R, Santoro N, Utian W et al. Stages of
reproductive aging workshop (STRAW). J Womens Health Gend Based Med 2001;
10: 843–848.
20 Garrett BE, Dube SR, Trosclair A, Caraballo RS, Pechacek TF. Cigarette smoking—
United States, 1965–2008. MMWR Surveill Summ 2011; 60: 109–113.
21 Lichtenstein AH, Appel LJ, Brands M, Carnethon M, Daniels S, Franch HA et al.
Diet and lifestyle recommendations revision 2006A scientific statement
from the American Heart Association nutrition committee. Circulation 2006; 114:
82–96.
22 World Health Organization Obesity: Preventing and Managing the GLOBAL Epi-
demic: Report of a WHO Consultation on Obesity, Geneva, 3–5 June 1997. World
Health Organization: Geneva, Switzerland, 1998. Report no. WHO/NUT/NCD/98.1.
23 Lipschitz DA. Screening for nutritional status in the elderly. Prim Care 1994; 21:
55–67.
24 Lohman TGAdvances in Body Composition Assessment. Human Kinetics Publishers:
Champaigne, IL, USAChampaigne, IL, USA1992 pp 80.
25 Antolovich M, Prenzler PD, Patsalides E, McDonald S, Robards K. Methods for
testing antioxidant activity. Analyst 2002; 127: 183–198.
26 Faulin S, do Espirito T, de Sena KCM, Rodrigues Telles AE, de Mattos Grosso D,
Bernardi Faulin EJ et al. Validation of a novel ELISA for measurement of electro-
negative low-density lipoprotein. Clin Chem Lab Med 2008; 46: 1769–1775.
27 Santo Faulin TdE, de Sena-Evangelista KCM, Pacheco DB, Augusto EM, Abdalla
DSP. Development of immunoassays for anti-electronegative LDL autoantibodies
and immune complexes. Clin Chim Acta 2012; 413: 291–297.
© 2015 Macmillan Publishers Limited
Oxidative stress biomarkers in breast cancer
AAF Carioca et al
1261
28 Okoh V, Deoraj A, Roy D. Estrogen-induced reactive oxygen species-mediated
signalings contribute to breast cancer. Biochim Biophys Acta 2011; 1815: 115–133.
29 Fussell KC, Udasin RG, Smith PJ, Gallo MA, Laskin JD. Catechol metabolites of
endogenous estrogens induce redox cycling and generate reactive oxygen spe-
cies in breast epithelial cells. Carcinogenesis 2011; 32: 1285–1293.
30 Žitnanová I, Rakovan M, Paduchová Z, Dvoráková M, Andrezálová L, Muchová J
et al. Oxidative stress in women with perimenopausal symptoms. Menopause
2011; 18: 1249–1255.
31 Sánchez-Rodríguez MA, Zacarías-Flores M, Arronte-Rosales A, Correa-Muñoz E,
Mendoza-Núñez VM. Menopause as risk factor for oxidative stress. Menopause
2012; 19: 361–367.
32 Pande D, Karki K, Negi R, Khanna S, Khanna RS, Khanna H. NF-κB p65 subunit
DNA-binding activity: association with depleted antioxidant levels in breast car-
cinoma patients. Cell Biochem Biophys 2013; 67: 1–7.
33 Şener DE, Gönenç A, Akıncı M, Torun M. Lipid peroxidation and total antioxidant
status in patients with breast cancer. Cell Biochem Funct 2007; 25: 377–382.
34 Silva IT, Mello A, Sanches L, Abdalla D, Damasceno N. Is plasma alpha-tocopherol
associated with electronegative LDL in obese adolescents? J Nutr Sci Vitaminol
2012; 59: 100–107.
35 Mafra D, Santos FR, Lobo JC, de Mattos Grosso D, Barreira AL, LGC Velarde et al.
Alpha-tocopherol supplementation decreases electronegative low-density lipo-
protein concentration [LDL ( − )] in haemodialysis patients. Nephrol Dial Transplant
2009; 24: 1587–1592.
36 Pereira EC, Bertolami MC, Faludi AA, Sevanian A, Abdalla DS. Antioxidant effect of
simvastatin is not enhanced by its association with α-tocopherol in hypercho-
lesterolemic patients. Free Radic Biol Med 2004; 37: 1440–1448.
37 Delimaris I, Faviou E, Antonakos G, Stathopoulou E, Zachari A, Dionyssiou-Asteriou
A. Oxidized LDL, serum oxidizability and serum lipid levels in patients with breast
or ovarian cancer. Clin Biochem 2007; 40: 1129–1134.
38 Khaidakov M, Mehta JL. Oxidized LDL triggers pro-oncogenic signaling in human
breast mammary epithelial cells partly via stimulation of MiR-21. PLoS One 2012; 7:
e46973.
39 Avogaro P, Bon GB, Cazzolato G. Presence of a modified low density lipoprotein
in humans. Arteriosclerosis 1988; 8: 79–87.
40 Estruch M, Sánchez-Quesada JL, Ordóñez Llanos J, Benítez S. Electronegative LDL:
a circulating modified LDL with a role in inflammation. Mediators Inflamm 2013;
2013: 181324.
41 Mello APQ, da Silva IT, Abdalla DSP, Damasceno NRT. Electronegative low-density
lipoprotein: origin and impact on health and disease. Atherosclerosis 2011; 215:
257–265.
42 Macciò A, Madeddu C. Obesity, inflammation, and postmenopausal breast cancer:
therapeutic implications. Sci World J 2011; 11: 2020–2036.
43 Subar AF, Kipnis V, Troiano RP, Midthune D, Schoeller DA, Bingham S et al. Using
intake biomarkers to evaluate the extent of dietary misreporting in a large sample
of adults: the OPEN study. Am J Epidemiol 2003; 158: 1–13.
44 Jenab M, Slimani N, Bictash M, Ferrari P, Bingham S. Biomarkers in nutritional
epidemiology: applications, needs and new horizons. Hum Genet 2009; 125:
507–525.
45 Pollard J, Wild CP, White KL, Greenwood DC, Cade JE, Kirk SF. Comparison of
plasma biomarkers with dietary assessment methods for fruit and
vegetable intake. Eur J Clin Nutr 2003; 57: 988–998.
46 Keogh RH, White IR, Rodwell SA. Using surrogate biomarkers to improve mea-
surement error models in nutritional epidemiology. Stat Med 2013; 32:
3838–3861.
47 Hodge AM, Simpson JA, Fridman M, Rowley K, English DR, Giles GG et al. Eva-
luation of an FFQ for assessment of antioxidant intake using plasma biomarkers in
an ethnically diverse population. Public Health Nutr 2009; 12: 2438–2447.
48 Wang Y, Yang M, Lee SG, Davis CG, Koo SI, Chun OK. Dietary total antioxidant
capacity is associated with diet and plasma antioxidant status in healthy
young adults. J Acad Nutr Diet 2012; 112: 1626–1635.
49 Frankenfeld CL, Lampe JW, Shannon J, Gao DL, Li W, Ray RM et al. Fruit and
vegetable intakes in relation to plasma nutrient concentrations in women in
ShanghaiPublic Health Nutr 2012; 15: 167–175.
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