- Email: [email protected]
Dietary antioxidant capacity and risk for stroke in a prospective cohort study of Swedish men and women
Accepted Manuscript Dietary Antioxidant Capacity and Risk of Stroke in a prospective cohort study of Swedish Men and Women Luca Colarusso, MSc, Mauro Serafini, PhD, Ylva Trolle Lagerros, MD PhD, Olof Nyren, MD PhD, Carlo La Vecchia, MD, Marta Rossi, ScD, Weimin Ye, MD PhD, Alessandra Tavani, ScD, Hans-Olov Adami, MD PhD, Alessandra Grotta, PhD, Rino Bellocco, ScD PII:
S0899-9007(16)30129-0
DOI:
10.1016/j.nut.2016.07.009
Reference:
NUT 9805
To appear in:
Nutrition
Received Date: 8 April 2016 Revised Date:
11 July 2016
Accepted Date: 16 July 2016
Please cite this article as: Colarusso L, Serafini M, Lagerros YT, Nyren O, La Vecchia C, Rossi M, Ye W, Tavani A, Adami H-O, Grotta A, Bellocco R, Dietary Antioxidant Capacity and Risk of Stroke in a prospective cohort study of Swedish Men and Women, Nutrition (2016), doi: 10.1016/j.nut.2016.07.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Dietary Antioxidant Capacity and Risk of Stroke in a prospective cohort study of
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Swedish Men and Women
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Luca Colarusso MSc*1, Mauro Serafini PhD*2, Ylva Trolle Lagerros MD PhD3,4, Olof
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Nyren MD PhD5, Carlo La Vecchia MD6, Marta Rossi ScD7,8, Weimin Ye MD PhD5,
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Alessandra Tavani ScD7, Hans-Olov Adami MD PhD5,8, Alessandra Grotta* PhD5,
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Rino Bellocco* ScD5,9.
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1
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Department of Statistics and Quantitative Methods, University of Milano-Bicocca, Milan, Italy. 2
Functional food and metabolic stress prevention laboratory, Center for Food and Nutrition, Council for Agricultural Research and Economics CREA, Rome, Italy. 3
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Department of Medicine, Clinical Epidemiology Unit, Karolinska Institutet, Stockholm, Sweden. 4
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Department of Medicine, Clinic of Endocrinology, Metabolism and Diabetes, Karolinska University Hospital Huddinge, Stockhlom, Sweden. 5
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. 6
Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy. Department of Epidemiology, Mario Negri Institute, Milan, Italy.
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Department of Epidemiology, Harvard School of Public Health, Boston MA, USA.
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Department of Statistics and Quantitative Methods, University of Milano-Bicocca, Milan, Italy.
These authors contributed equally to the manuscript.
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Correspondence to: Rino Bellocco, Sc.D., Department of Statistics and Quantitative Methods, University of Milano-Bicocca, Milan, Italy.
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E-mail [email protected]; phone number: +39 02 64485831; fax number: +39
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02 64485899
ACCEPTED MANUSCRIPT Abstract
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Objective: Both observational studies and randomized trials have shown that a diet
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rich in antioxidants can reduce systemic inflammation and oxidative stress, two
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conditions that, together with obesity and smoking, are established risk factors for
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stroke. However, the association between antioxidant intake and risk of stroke is
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poorly understood, particularly when studying possible interaction with gender. We
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investigated the relationship of Non Enzymatic Antioxidant Capacity (NEAC) on risk
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of stroke in a large Swedish prospective cohort.
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Methods: This cohort study included 34,555 men and women from the Swedish
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National March Cohort. NEAC was assessed using a detailed food frequency
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questionnaire, collected at baseline. We achieved complete follow-up from enrolment
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in 1997 through 2010 by record linkage to nation-wide registers.
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We identified 1,186 incident cases of a first stroke, of which 860 were ischemic, 201
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hemorrhagic and 125 unspecified. We used multivariable Cox proportional hazards
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models to estimate adjusted hazard ratios (HR) with 95% confidence intervals (CI).
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Results: Compared to women in the lowest quartile of NEAC women in the highest
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quartile had a 27% lower incidence of total stroke (HR=0.73; 95% CI: 0.53-0.99; p
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for trend = 0.03) and 35% lower incidence of ischemic stroke (HR=0.65; 95% CI:
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0.43-0.99; p for trend =0.01). Among men, the relationship between NEAC and risk
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of stroke was not statistically significant and all HRs were close to unity.
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Conclusion: Our findings suggest that dietary antioxidant capacity from different
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Atherosclerosis; Risk Factors; Cohort Studies.
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foods and beverages is inversely associated with risk of stroke, more specifically ischemic stroke, in women. Keywords: Stroke; Antioxidant Effect; Dietary Habits; Epidemiology;
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ACCEPTED MANUSCRIPT Introduction
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Stroke is the second leading cause of death in the world [1]. Well-known modifiable
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risk factors for this neurovascular emergency include hypertension, obesity, physical
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inactivity, an unhealthy diet and smoking [2]. Furthermore, oxidative and
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inflammatory stress promotes atherosclerosis [3] that ultimately can lead to stroke [2,
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4]. A high consumption of flavonoids and phytochemicals with antioxidant properties
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contained in fruits and vegetables is associated with reduced oxidative stress [5] and
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reduced systemic inflammation [6, 7]. Antioxidants in fruits, vegetables, and other
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foods and beverages may therefore prevent stroke and other cardiovascular diseases
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by reducing excessive production of free radicals induced by oxidative and
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inflammatory stress [8-15].
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However, few observational studies have investigated the association between total
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antioxidant capacity from diet and risk of stroke [10, 11, 16]. Two studies found an
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inverse association between consumption of antioxidants and risk of stroke. A third
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study did not find any evidence of neither a positive, nor a negative association [16].
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Therefore, the association remains open for discussion. Moreover, a possible
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interaction with gender has not been investigated yet. However, previous studies have
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suggested a possible gender difference in antioxidant status [17], which may in turn
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lead to a potential different effect of dietary antioxidants on the risk of stroke.
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capacity assays such as ferric reducing antioxidant potential (FRAP) [20].
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Non Enzymatic Antioxidant Capacity (NEAC), also known as total antioxidant capacity (TAC), is a measure proposed to assess the cumulative power offered by all antioxidant sources from diet [18, 19]. NEAC can be estimated through antioxidant
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We investigated the relationship of NEAC, estimated using food item-specific
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antioxidant FRAP values, on risk of stroke in a large Swedish prospective cohort of
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women and men.
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Methods
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The Swedish National March Cohort (SNMC) is a prospective study established in
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Sweden in 1997 during a four-day national fund-raising event promoted by the
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Swedish Cancer Society. Participants in the SNMC were invited to fill out a 36-page
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questionnaire concerning socio-demography, lifestyle, diet and medical history at
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baseline. An 85-item food frequency questionnaire (FFQ), a slightly abbreviated
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version of a validated 96-item questionnaire [21], was used to estimate individual
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intake of common Swedish food items. The participants were asked to indicate how
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often, on average in the previous year, they had consumed these foods and beverages.
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Eight response categories ranged from “never/seldom” to “3 or more times per day”.
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We computed body mass index (BMI) by dividing reported weight (kg) by the
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squared height (m) and categorized it according to the World Health Organization
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classification (underweight: <18.5; normal weight: 18.5 to < 25 kg/m2; overweight: 25
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to < 30 kg/m2; obese: ≥ 30 kg/m2). Physical activity was assessed by a validated
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questionnaire [22]; time spent on each of nine predefined intensity levels of physical
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activity during a typical weekday was self-estimated, and the estimates were
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multiplied by their respective metabolic equivalent (MET) values [22]. Finally, the products were summed up and total physical activity was reported in terms of METh/day.
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Food item-specific antioxidant capacity values were obtained from a database with
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“in vitro” measurements using the FRAP assay [20, 23]. A total of 66 out of the
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original 85 FFQ items had FRAP values in the database and were used for dietary
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NEAC computation. These values were multiplied by the reported frequency of
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consumption taking the portion size into account. Coffee consumption was left out
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when computing NEAC due to the observed discrepancy between the “in vivo” and
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“in vitro” effects of the Maillard products [24, 25]. Other foods, such as chocolate and
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whole grains, were not excluded in the antioxidant computation since they have a
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lower content of melanoidins. Moreover, these foods have a high level of other
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bioactive ingredients such as flavonoids. We have followed the same approach in a
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previous study [26]. Since FRAP values for dietary supplements were not available,
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antioxidant supplements were left out of NEAC computation.
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The reproducibility and validity of NEAC values derived from food frequency
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questionnaire data combined with FRAP assays are reported in the paper from
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Rautiainen et al. [21]; the correlation between NEAC was adjusted for total energy
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intake using the residual method [27], since estimated NEAC correlated with total
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energy intake (ρ=0.4; p <0.05). The validity of the dietary food items contributing to
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NEAC compared to food record was acceptably good in a similar Swedish cohort.
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The estimated Pearson correlation coefficients ranged from 0.4 to 0.8 (A. Wolk,
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unpublished data).
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Follow-up
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ACCEPTED MANUSCRIPT A total of 43,863 men and women completed the questionnaire and consented to
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follow-up through record linkages with multiple national registries using the
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individually unique national registration numbers to ensure perfect matching. We
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excluded from the analyses 1,740 participants under the age of 18, 19 participants
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with invalid national registration number or conflicting answers, 457 who were
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recorded as emigrated before the start of follow-up, and 4,045 with any recorded
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cardiovascular diagnosis (e.g., stroke, myocardial infarction, heart failure, angina
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pectoris, and atrial fibrillation; International Classification of Diseases, [ICD7] 330-
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334, 400-468, [ICD8] 390-458, [ICD9] 390–459, and [ICD10] I00-I99 before the start
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of follow-up. We also excluded 2,652 participants with a previous history of cancer
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recorded in the Swedish Cancer Register (except non-melanoma skin cancer), and 406
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participants with extreme values for total energy intake (± 3 standard deviations for
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the mean value of loge transformed energy, ≤927 or >5,311 kcal for men, and ≤870 or
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>4,348 kcal for women). The remaining 34,555 participants were followed from
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October 1, 1997 until time of first hospitalization for stroke, emigration, death, or
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December 31, 2010, whichever occurred first. Incident stroke cases were identified in
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the nationwide and essentially complete registers of Inpatient Care and Causes of
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Death using the following ICD10 codes: I63.0-I63.5, I63.8-I63.9 (cerebral infarction);
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I61 (intracerebral haemorrhage); I60 (subarachnoid haemorrhage); I64 (unspecified
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stroke).
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variable. Descriptive statistics were presented for the whole cohort according to sex-
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specific quartiles of NEAC intake. We used Cox proportional hazards regression
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Statistical Analysis
Dietary NEAC values were categorized in quartiles, but also analyzed as a continuous
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ACCEPTED MANUSCRIPT models to assess the association between dietary NEAC intake and stroke risk. Hazard
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ratios (HRs) and the corresponding 95% confidence intervals (CIs) were obtained
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using age as the underlying time scale; they were calculated for categorical NEAC
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sex-specific quartiles using the lowest quartile as the reference category. The
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multivariable models were adjusted for the following potential confounders: sex, BMI
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(<18.5, 18.5-24.9, 25.0-29.9, 30+ kg/m2), alcohol consumption (never, low: ≤1 times
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per week, medium: >1 to 6 times per week and, high: ≥7 times per week, based on the
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frequency of drinking), smoking status (never, former and current smokers), physical
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activity (METh/day), educational level (<12 years or ≥12 years), dietary supplement
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use (yes/no), self-reported hypertension (yes/no), self-reported diabetes (yes/no),
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coffee consumption (daily consumption in quartiles), aspirin use (yes/no), self-
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reported lipid disturbance (yes/no) and, only for women, hormone replacement
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therapy use (yes/no). Moreover, we repeated our analyses by adjusting for self-
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reported menopausal status. To assess the proportional hazards assumption, we used
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the deviance test based on scaled Schoenfeld residuals. The proportional hazards
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assumption was satisfied in almost all multivariable models; when this assumption
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was not fulfilled, stratified Cox regression models were fitted.
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We assessed a linear trend across quartiles of NEAC using the median value of each
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NEAC category and including the resulting series of values in the models as a
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continuous variable. The validity of the assumption of a linear relationship between
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subgroup analyses according to sex, age (≤60, >60 years), BMI (<25, ≥25 kg/m2),
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NEAC and the stroke incidence was evaluated in Cox regression by adding a quadratic term and testing its significance. We also used a restricted cubic spline with 3 knots corresponding to the 5th, 50th and 75th percentiles [28]. We performed
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hypertension (yes/no), diabetes (yes/no), smoking (yes/no) and dietary supplement
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use (yes/no). We tested for statistical significance of effect modification on a
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multiplicative scale. Deviation from multiplicativity was evaluated using the
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likelihood ratio (LRT) test to compare nested models.
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The proportion of missing data on covariates were the following: 11.9% for total
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physical activity, 7.7% for smoking status, 4.9% for self-reported diabetes, 4.9% for
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BMI, 4.2% for self-reported lipid disturbance, 3.2% for self-reported hypertension;
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for the other covariates the proportion of missing information was below 2%.
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We used multiple imputation techniques to assess the robustness of estimates based
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on complete data to the presence of missing values. Under the assumption of data
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missing at random, we fitted multiple imputation models based on chained equations
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[29, 30], generating five imputed datasets from the initial cohort. HRs from the
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imputed datasets were combined using Rubin’s rules producing the pool effect
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estimate and relative estimated standard errors.
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To investigate the possible impact of reverse causality on the results we excluded
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cases during the first 3 years of follow-up to account for changes in dietary habits due
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to undiagnosed cardiovascular disease. We also repeated our main analyses using
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calendar time as time scale and adjusting for age.
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All probability values were presented as two-sided and p-values < 0.05 were
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considered statistically significant. All statistical analyses were performed using Stata
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(Version 13.1; StataCorp LP). 8
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The Regional Ethics Review Board at the Karolinska Institutet approved the study.
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Results
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Table 1 shows baseline characteristics of the cohort according to sex-specific quartiles
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of NEAC. Median NEAC intake was slightly lower in men compared to women. The
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major contribution to NEAC came from fruit and vegetable consumption (27.6% in
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women, 22.5% in men), tea (29.0% in women, 24.0% in men), whole grains (8.7% in
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women, 11.3% in men) and chocolate (9.3% in women and 10.3% in men). Men and
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women in the highest quartile of dietary NEAC were on average older than those in
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the lowest quartile. They were also more likely to use dietary supplements, to
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consume more fruits and vegetables, less coffee, to be non-smokers, and to have a
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higher education.
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During 438, 272 person-years of follow-up 1,186 participants had a first stroke (657
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women, 529 men). Of these stroke events, 860 were ischemic, 201 hemorrhagic and
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125 unspecified. Table 2 and Table 3 report results from separate Cox regression
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models for each of the subtypes as well as total stroke, adjusted for age and potential
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confounders and stratified according to sex (p for interaction on the multiplicative
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scale between sex and NEAC for total stroke=0.02). Among women, an inverse
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association between NEAC intake and total stroke incidence was found. Females in the highest quartile of NEAC had a 27% (95% CI: 0.53-0.99) lower incidence of total
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stroke, compared with those in the lowest quartile with a statistically significant linear
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trend in risk (p for trend=0.03). The observed inverse association with NEAC intake 9
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in women was driven mainly by the trend observed for ischemic stroke. Women with
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the highest NEAC intake had a 35% lower incidence of ischemic stroke (95% CI:
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0.43-0.99; p for trend=0.01) while no clear associations were found with hemorrhagic
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stroke or unspecified stroke, admittedly based on small numbers. After adjusting for
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menopausal status, results did not change substantially. Among men, we were unable
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to confirm any association between dietary NEAC and stroke of any type (Table 3).
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The association between NEAC and risk of stroke in women did not change
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significantly by age, history of diabetes, hypertension, alcohol consumption, and
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dietary supplement use.
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When re-running the same statistical analyses to assess the potential impact of the
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missingness on the observed associations, HRs based on multiple imputed datasets
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confirmed findings from complete data analysis (data not shown). In addition, we
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observed similar patterns when the exposure was treated as a continuous variable.
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Finally, when we excluded cases occurring in the first 3 years of follow-up, results did
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not change materially: women in the highest, as compared with the lowest quartile of
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NEAC intake had a 27% and 34% lower hazard of total stroke (HR=0.73; 95% CI:
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0.53-1.00; p for trend: 0.05) and ischemic stroke (HR=0.66; 95% CI: 0.39-0.98; p for
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trend=0.04), respectively. We observed the same results when using calendar time as
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time scale and adjusting for age at recruitment.
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Discussion
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In this large prospective study, dietary NEAC intake was associated with a reduced
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incidence of stroke, more specifically ischemic stroke, in women. Compared with
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women in the lowest quartile of dietary NEAC, those in the highest quartile had a
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35% lower incidence of ischemic stroke, and a 27% lower incidence of total stroke.
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No consistent association was found in men.
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Epidemiological studies have found an inverse association between risk of stroke and
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single food items such as tea [31], chocolate [32, 33], fruits and vegetables [8] and
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cereals [34-36]. Further, randomized intervention studies [37, 38] have highlighted
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the role of antioxidants from diet in preventing or mitigating systemic inflammation,
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oxidative stress and atherosclerosis.
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Therefore, the concept that dietary antioxidant capacity may play a role in stroke
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incidence is not completely new. However, to our knowledge, only three previous
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studies [10, 11, 16] investigated the association between the overall antioxidant
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capacity, rather than individual contributions from single food items, and the risk of
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stroke.
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Our results are consistent with two of these studies showing a marginally significant
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inverse association between NEAC and the risk of stroke [10, 11]. The first one, the
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that a high NEAC intake was inversely associated with total stroke among women
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Italian section of the European Prospective Investigation into Cancer and Nutrition (EPIC), found that dietary NEAC was inversely related to ischemic stroke, but not with hemorrhagic stroke. The second one, the Swedish Mammography Cohort, found
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quartile of NEAC (HR=0.83; 95% CI: 0.70-0.95). Our data suggest a role of NEAC in
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preventing stroke in women. A greater role of dietary NEAC on the relative risk of
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stroke in women compared to men is conceivable, considering the difference in
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background incidence, and the higher levels of NEAC in women’s diet. An unclear
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increase in the incidence of ischemic stroke is found among men with the highest
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NEAC intake. This result is not straightforward to interpret and indicates the need for
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further studies investigating different effects of NEAC by gender.
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The stronger association found with ischemic rather than hemorrhagic stroke is not
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surprising, since diet and lifestyle have been shown to be stronger associated with
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ischemic than hemorrhagic stroke [15, 39, 40]. The reduction in stroke incidence
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observed among women with a high dietary antioxidant intake can be related to the
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antioxidants’ ability to counteract the circulating levels of free radicals that are
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involved in the systemic inflammation and oxidative stress. Another important aspect
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to take into account involves the homeostatic redox process and its balancing
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mechanism between endogenous and exogenous antioxidants. When oxidative stress
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is absent, endogenous defenses are sufficient to hinder the production of free radicals.
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But when oxidative stress is ongoing, endogenous antioxidants are not able to inhibit
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the production of free radicals efficiently. Therefore, the contribution of exogenous
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antioxidants from diet may be crucial to support the endogenous redox machinery.
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A third study, the Rotterdam Study [16] did not find an association between NEAC intake and risk of stroke. This may depend on several factors, including a smaller
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sample size and different exclusion criteria, other major sources of NEAC, and a
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higher mean age.
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The fact that the consumption of the most important dietary sources of antioxidants in
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our study (fruits and vegetables, and tea) was lower among men than among women
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reduces our power to detect an association. When considering the 19 food items not
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included in the NEAC computation, there were no particular differences between men
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and women in the food frequency consumption that could somewhat explain the
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observed association among women, but not among men.
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NEAC computation originates from the hypothesis that antioxidants from foods may
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work as antioxidants in vivo, which is an extremely complex aspect of the dietary
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modulation of oxidative stress and disease prevention; measurements of biological
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biomarkers would be needed in order to better understand the mechanism. However,
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we believe that in vitro NEAC estimates can be useful to guide researchers to better
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understand the antioxidant effect on humans, but further studies on plasma NEAC
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could help to understand the real mechanism of antioxidant action.
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Our study has several strengths. The most important one is the use of NEAC as a
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measure of the overall antioxidant potential from the diet. Other important strengths
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are the prospective design, the detailed data on diet and potential risk factors and the
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large sample size. Furthermore the large number of stroke cases and record linkage to
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nation-wide registers allowed us for a nearly complete follow-up of study outcomes.
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information on bioavailability. Foods with the highest content of antioxidants may not
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The most important limitation is due to the fact that, although dietary assessment of NEAC is correlated with plasma antioxidant levels [21], it does not provide direct
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ACCEPTED MANUSCRIPT necessarily be those leading to the highest concentrations of active metabolites in
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vivo. In fact, the use of a coefficient of absorption based on previous evidences from
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ingestion studies, has recently been proposed in the literature [12], but this approach
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could be questionable. Another limitation is the use of only one semi-quantitative
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food frequency questionnaire which allow assessment of the frequencies of
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consumption, but not the exact amount of food consumption, and it does not consider
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the use of antioxidant supplements. Finally, the use of FRAP values calculated from a
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non-Swedish food database items [20, 23]. It should be noticed that geographic
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location and growing conditions might not entirely explain the observed within study
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variability and therefore, using antioxidant values from countries with eating habits
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similar to those in Sweden may not be more appropriate than using an Italian food
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items database.
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Another limitation could be represented by the fact that FRAP values for dietary
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supplements were not available and our results cannot be related to antioxidant
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supplements, but only to food items.
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Conclusion
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In conclusion, this study shows that a high-NEAC diet is associated with a reduced
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risk of ischemic (and total) stroke in women, and suggests a potential role of a diet
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rich in fruit, vegetables and, food with antioxidant properties in the prevention of the
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disease.
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Acknowledgments
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This work was conducted with the contribution of the Italian Ministry of University
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and Research (PRIN 2009 X8YCBN), the Swedish Cancer Society (Grant CAN 14
ACCEPTED MANUSCRIPT 2012/591), Karolinska Institutet Distinguished Professor Award to Hans-Olov Adami
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(Dnr: 2368/10-221); and the regional agreement on medical training and clinical
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research between Stockholm County Council and Karolinska Institutet for Ylva Trolle
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Lagerros. The authors would like to thank Keith Humpreys (Associate Professor of
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Biostatistics, Department of Medical Epidemiology and Biostatistics, Karolinska
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Institutet) for language editing.
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Conflict of Interest: none.
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[13] Rautiainen S, Levitan EB, Mittleman MA, Wolk A. Total antioxidant capacity of diet and risk of heart failure: a population-based prospective cohort of women. American Journal of Medicine. 2013;126:494-500. [14] Kim K, Vance TM, Chun OK. Greater Total Antioxidant Capacity from Diet and Supplements Is Associated with a Less Atherogenic Blood Profile in U.S. Adults. Nutrients. 2016;8. [15] Rossi M, Praud D, Monzio Compagnoni M, Bellocco R, Serafini M, Parpinel M, et al. Dietary non-enzymatic antioxidant capacity and the risk of myocardial infarction: a casecontrol study in Italy. Nutrition, Metabolism and Cardiovascular Diseases. 2014;24:1246-51. [16] Devore EE, Feskens E, Ikram MA, den Heijer T, Vernooij M, van der Lijn F, et al. Total antioxidant capacity of the diet and major neurologic outcomes in older adults. Neurology. 2013;80:904-10. [17] Brunelli E, Domanico F, La Russa D, Pellegrino D. Sex differences in oxidative stress biomarkers. Current Drug Targets. 2014;15:811-5. [18] Serafini M, Del Rio D. Understanding the association between dietary antioxidants, redox status and disease: is the Total Antioxidant Capacity the right tool? Redox Report 2004;9:145-52. [19] Serafini M, Miglio C, Peluso I, Petrosino T. Modulation of plasma non enzimatic antioxidant capacity (NEAC) by plant foods: the role of polyphenols. Current Topics in Medicinal Chemistry. 2011;11:1821-46. [20] Pellegrini N, Serafini M, Colombi B, Del Rio D, Salvatore S, Bianchi M, et al. Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. Journal of Nutrition. 2003;133:2812-9. [21] Rautiainen S, Serafini M, Morgenstern R, Prior RL, Wolk A. The validity and reproducibility of food-frequency questionnaire-based total antioxidant capacity estimates in Swedish women. American Journal of Clinical Nutrition 2008;87:1247-53. [22] Ainsworth BE, Haskell WL, Herrmann SD, Meckes N, Bassett DR, Jr., Tudor-Locke C, et al. 2011 Compendium of Physical Activities: a second update of codes and MET values. Medicine and science in sports and exercise. 2011;43:1575-81. [23] Pellegrini N, Serafini M, Salvatore S, Del Rio D, Bianchi M, Brighenti F. Total antioxidant capacity of spices, dried fruits, nuts, pulses, cereals and sweets consumed in Italy assessed by three different in vitro assays. Molecular nutrition & food research. 2006;50:1030-8. [24] Delgado-Andrade C MFJ. Unraveling the contribution of melanoidins to the antioxidant activity of coffee brews. Journal of Agricultural and Food Chemistry 2005:1403-7. [25] Morales FJ, Somoza V, Fogliano V. Physiological relevance of dietary melanoidins. Amino Acids. 2012;42:1097-109. [26] Serafini M, Jakszyn P, Lujan-Barroso L, Agudo A, Bas Bueno-de-Mesquita H, van Duijnhoven FJ, et al. Dietary total antioxidant capacity and gastric cancer risk in the European prospective investigation into cancer and nutrition study. International Journal of Cancer. 2012;131:E544-54. [27] Willett WC, Howe GR, Kushi LH. Adjustment for total energy intake in epidemiologic studies. American Journal of Clinical Nutrition. 1997;65:1220S-8S; discussion 9S-31S. [28] Harrell FE, Lee KL, Pollock BG. Regression models in clinical studies: determining relationships between predictors and response. Journal of the National Cancer Institute. 1988;80:1198-202. [29] Royston P. Multiple imputation of missing values. Stata Journal. 2004;4:227-41. [30] van Buuren S, Boshuizen HC, Knook DL. Multiple imputation of missing blood pressure covariates in survival analysis. Statistics in Medicine. 1999;18:681-94. [31] Arab L, Liu W, Elashoff D. Green and black tea consumption and risk of stroke: a metaanalysis. Stroke. 2009;40:1786-92.
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[32] Buijsse B, Weikert C, Drogan D, Bergmann M, Boeing H. Chocolate consumption in relation to blood pressure and risk of cardiovascular disease in German adults. European Heart Journal. 2010;31:1616-23. [33] Mink PJ, Scrafford CG, Barraj LM, Harnack L, Hong C-P, Nettleton JA, et al. Flavonoid intake and cardiovascular disease mortality: a prospective study in postmenopausal women. American Journal of Clinical Nutrition. 2007;85:895-909. [34] Ascherio A, Rimm EB, Hernan MA, Giovannucci EL, Kawachi I, Stampfer MJ, et al. Intake of potassium, magnesium, calcium, and fiber and risk of stroke among US men. Circulation. 1998;98:1198-204. [35] Larsson SC, Männistö S, Virtanen MJ, Kontto J, Albanes D, Virtamo J. Dietary fiber and fiber-rich food intake in relation to risk of stroke in male smokers. European Journal of Clinical Nutrition 2009;63:1016-24. [36] Oh K, Hu FB, Cho E, Rexrode KM, Stampfer MJ, Manson JE, et al. Carbohydrate intake, glycemic index, glycemic load, and dietary fiber in relation to risk of stroke in women. American Journal of Epidemiology. 2005;161:161-9. [37] Brighenti F, Valtuena S, Pellegrini N, Ardigo D, Del Rio D, Salvatore S, et al. Total antioxidant capacity of the diet is inversely and independently related to plasma concentration of high-sensitivity C-reactive protein in adult Italian subjects. British Journal of Nutrition. 2005;93:619-25. [38] Valtueña S, Pellegrini N, Franzini L, Bianchi MA, Ardigò D, Del Rio D, et al. Food selection based on total antioxidant capacity can modify antioxidant intake, systemic inflammation, and liver function without altering markers of oxidative stress. American Journal of Clinical Nutrition. 2008;87:1290-7. [39] Misirli G, Benetou V, Lagiou P, Bamia C, Trichopoulos D, Trichopoulou A. Relation of the traditional mediterranean diet to cerebrovascular disease in a mediterranean population. American Journal of Epidemiology. 2012;176:1185-92. [40] Miglio C, Peluso I, Raguzzini A, Villaño DV, Cesqui E, Catasta G, et al. Fruit juice drinks prevent endogenous antioxidant response to high-fat meal ingestion. British Journal of Nutrition. 2013:1-7.
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Table 1: Age-standardized baseline characteristics of participants in the Swedish
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National March Cohort: A) women B) men
Women (n=22,712)
RI PT
Non-Enzymatic Antioxidant Capacity* Q1 (n=5,678)
Q2 (n=5,678)
Q3 (n=5,678)
Q4 (n=5,678)
5.8 (5.1-6.6)
8.3 (7.8-8.9)
10.8 (10.1-11.6)
15.2 (13.6-17.9)
Age, years, mean(SD)
48.0 (15.0)
48.9 (15.2)
48.8 (15.2)
49.7 (15.0)
>12 years of education,
36.0
42.3
46.9
55.8
8.5
7.6
6.1
24.6 (3.8)
24.3 (3.7)
24.1 (3.5)
23.9 (3.5)
TE
(A)
37.5 (11.6)
37.7 (11.6)
37.7 (11.9)
Median NEAC (IQR)
SC
(mmol/d)
% 11.6
D
Current smokers, % Body Mass Index, mean kg/m2(SD)
37.2 (11.9)
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Total Physical Activity
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Baseline characteristics
(SD)
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Score (MET*h/d), mean
Aspirin use, %
65.4
66.2
65.9
64.5
Dietary supplement use,
50.4
53.1
55.1
57.0
%
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ACCEPTED MANUSCRIPT Self-reported
10.7
10.4
9.9
11.0
Self-reported diabetes, %
1.7
1.7
1.8
2.0
Self-reported lipid
2.8
2.3
2.6
2.2
0.7
1.0
1.1
1.7
24.9
25.8
1948.8 (496.4)
2048.0 (496.3)
hypertension, %
Alcohol consumption
RI PT
disturbance, %
(high), % Hormone replacement
27.0
Total energy intake
SC
use %
3.6 (1.7)
4.6 (2.0)
5.2 (2.4)
5.6 (2.9)
1.2 (0.4)
1.3 (0.5)
1.4 (0.5)
1.4 (0.5)
1.3 (0.7)
1.8 (0.9)
3.1 (2.0)
Foods, mean (SD) Fruits and vegetables, servings/d
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Whole Grains,
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servings/d
1.0 (0.2)
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Tea, servings/d
1961.1 (511.7)
M AN U
(kcal/d), mean (SD)
2065.1 (500.0)
29.0
0.8 (0.7)
1.1 (1.1)
1.3 (1.5)
1.4 (2.0)
Coffee, servings/d
3.3 (1.6)
3.0 (1.5)
2.6 (1.4)
2.0 (1.6)
25.6 (8.7)
25.9 (8.5)
25.4 (8.4)
23.5 (8.5)
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Chocolate, servings/wk
Nutrients, mean(SD) Saturated fatty acids, gr/day
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ACCEPTED MANUSCRIPT Monounsaturated fatty
18.6 (5.8)
19.1 (5.9)
19.0 (5.9)
17.5 (5.8)
6.0 (2.0)
6.5 (2.0)
6.6 (2.1)
6.3 (2.1)
acids, gram/day Polyunsaturated fatty acids, gram/day
Non-Enzymatic Antioxidant Capacity*
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(B) Men (n=11,843)
Q1 (n=2,961)
Q2 (n=2,961)
Q3 (n=2,961)
Q4 (n=2,960)
Median NEAC (IQR)
5.4 (4.7-5.9)
7.4 (6.9-7.9)
9.4 (8.9-10.1)
13.2 (11.8-15.2)
SC
(mmol/d)
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Baseline characteristics Age, years, mean(SD)
46.8 (17.9)
49.5 (17.5)
51.7(16.7)
54.4 (15.1)
>12 years of education,
36.7
41.4
47.6
55.0
7.8
5.6
4.9
25.1 (3.2)
24.9 (3.3)
24.6 (3.2)
44.1 (17.5)
44.0 (15.9)
42.9 (15.3)
42.3 (15.4)
Aspirin use, %
60.8
60.9
61.3
58.9
Dietary supplement use,
30.7
35.5
40.0
43.0
%
EP
kg/m2(SD)
25.4 (3.3)
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Body Mass Index, mean
10.5
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Current smokers, %
Total Physical Activity
(SD)
AC C
Score (MET*h/d), mean
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ACCEPTED MANUSCRIPT % Self-reported
9.1
9.1
9.5
9.3
Self-reported diabetes, %
3.0
2.6
2.3
2.3
Self-reported lipid
2.2
2.3
2.6
2.5
2.6
2.2
2227.8 (616.0)
2344.5 (585.6)
hypertension, %
Alcohol consumption (high), %
(kcal/d), mean (SD)
Fruits and vegetables,
2.7 (1.4)
2358.3 (618.3)
2269.9 (653.9)
3.5 (1.7)
4.0 (2.0)
4.3 (2.3)
1.5 (0.5)
1.6 (0.5)
1.6 (0.6)
1.0 (0.2)
1.3 (0.5)
1.8 (0.7)
3.1 (1.5)
0.7 (0.7)
1.1 (1.1)
1.4 (1.5)
1.7 (2.2)
4.7 (1.8)
4.4 (1.8)
4.0 (1.8)
3.5 (2.0)
29.5 (10.7)
29.5 (9.8)
29.0 (10.3)
27.4 (10.6)
21.0 (6.9)
21.7 (6.6)
21.5 (6.9)
20.5 (7.3)
servings/d Whole Grains,
1.4 (0.5)
EP
TE
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servings/d
Chocolate, servings/wk
4.7
M AN U
Foods, mean (SD)
Tea, servings/d
3.5
SC
Total energy intake
RI PT
disturbance, %
Coffee, servings/d
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Nutrients, mean(SD) Saturated fatty acids, gr/day
Monounsaturated fatty
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ACCEPTED MANUSCRIPT acids, gram/day Polyunsaturated fatty
6.6 (2.3)
7.2 (2.3)
7.3 (2.5)
7.1 (2.6)
acids, gram/day *NEAC was estimated through ferric reducing antioxidant power (FRAP); Q, quartile; MET, metabolic equivalents.
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486
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Table 2: Dietary Non-enzymatic antioxidant capacity (NEAC) and risk of
489
stroke in the Swedish National March Cohort among women.
WOMEN
Non-Enzymatic Antioxidant Capacity* Q1
Q2
Q3
Q4
p for
Total Stroke
RI PT
trend
168
182
163
Person-years,
72,876
72,650
72,794
Age-adjusted HR
1.0
0.99 (0.81-1.22)
0.89 (0.72-1.11)
Multivariable HR†
1.0
0.99 (0.75-1.31)
No. of cases
116
Age-adjusted HR Multivariable HR†
0.93 (0.69-1.24)
0.73 (0.53-0.99)
0.03
144
108
99
1.0
1.13 (0.89-1.44)
0.85 (0.66-1.11)
0.73 (0.56-0.95)
<0.01
1.0
1.20 (0.85-1.68)
D
0.93 (0.65-1.34)
0.65 (0.43-0.99)
0.01
M AN U
<0.01
TE EP
No. of cases
72,652
0.74 (0.59-0.92)
Ischemic
Haemorrhagic
144
SC
No. of cases
34
24
38
29
1.0
0.67 (0.40-1.13)
1.01 (0.67-1.68)
0.77 (0.47-1.27)
0.61
1.0
0.58 (0.30-1.21)
1.1 (0.62-1.95)
0.85 (0.45-1.60)
0.97
No. of cases
18
14
17
16
Age-adjusted HR
1.0
0.71 (0.35-1.42)
0.86 (0.44-1.67)
0.74 (0.38-1.46)
0.53
Multivariable HR†
1.0
0.74 (0.32-1.72)
0.49 (0.19-1.29)
0.84 (0.35-2.02)
0.71
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Age-adjusted HR
Multivariable HR† Unspecified
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* NEAC was estimated through ferric reducing antioxidant power (FRAP); Q, quartile (Q1
491
reference).
492
† Adjusted for age, education level, smoking status, body mass index, physical activity, self-
493
reported hypertension, self-reported diabetes, aspirin use, dietary supplement use, coffee
494
consumption, alcohol consumption, self-reported lipid disturbance, and total energy intake.
495
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EP
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M AN U
SC
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496
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ACCEPTED MANUSCRIPT 497
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Table 3: Dietary Non Enzymatic Antioxidant Capacity (NEAC) and risk of stroke in the Swedish
499
National March Cohort among men.
Non-Enzymatic Antioxidant Capacity* Q1
RI PT
MEN
Q2
Q3
106
Person-years,
37,116
Age-adjusted HR
1.0
Multivariable HR†
1.0
trend
168
36,942
36,779
36,585
1.0 (0.77-1.29)
0.97 (0.75-1.25)
1.10 (0.87-1.41)
0.36
1.19 (0.85-1.67)
0.99 (0.70-1.40)
1.35 (0.96-1.89)
0.11
93
98
129
1.0
1.08 (0.80-1.47)
1.04 (0.77-1.41)
1.22 (0.92-1.63)
0.17
1.0
1.27 (0.86-1.88)
1.08 (0.73-1.61)
1.55 (1.07-2.33)
0.05
No. of cases
18
18
16
24
Age-adjusted HR
1.0
0.88 (0.46-1.70)
0.72 (0.37-1.41)
0.97 (0.53-1.79)
0.99
Multivariable HR†
1.0
1.0 (0.41-2.51)
0.74 (0.29-1.89)
0.91 (0.37-2.25)
0.81
EP
Age-adjusted HR
TE
73
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No. of cases
p for
131
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Ischemic
124
M AN U
No. of cases
SC
Total Stroke
Q4
Multivariable HR† Haemorrhagic
25
ACCEPTED MANUSCRIPT Unspecified No. of cases
15
13
17
15
Age-adjusted HR
1.0
0.73 (0.35-1.54)
0.88 (0.44-1.77)
0.69 (0.34-1.42)
0.43
Multivariable HR††
1.0
0.80 (0.29-2.24)
0.68 (0.24-1.95)
0.56 (0.18-1.76)
0.34
* NEAC was estimated through ferric reducing antioxidant power (FRAP); Q, quartile (Q1
501
reference).
502
†Adjusted for age, education level, smoking status, body mass index, physical activity, self-
503
reported hypertension, self-reported diabetes, aspirin use, dietary supplement use, coffee
504
consumption, alcohol consumption, self-reported lipid disturbance, and total energy intake.
505
†† Adjusted for age, education level, smoking status, body mass index, physical activity, self-
506
reported hypertension, self-reported diabetes, dietary supplement use, coffee consumption,
507
alcohol consumption, self-reported lipid disturbance, and total energy intake and stratified by
508
aspirin use because the proportionality assumption did not hold.
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513
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ACCEPTED MANUSCRIPT HIGHLIGHTS: Dietary NEAC is a measure of the overall antioxidant capacity of diet. Dietary NEAC takes into account synergistic interactions among dietary antioxidants. The relationship between dietary NEAC and risk of stroke has been not extensively explored. High levels of dietary NEAC are associated with a decreased risk of ischemic stroke among women.
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1. 2. 3. 4.
S0899-9007(16)30129-0
DOI:
10.1016/j.nut.2016.07.009
Reference:
NUT 9805
To appear in:
Nutrition
Received Date: 8 April 2016 Revised Date:
11 July 2016
Accepted Date: 16 July 2016
Please cite this article as: Colarusso L, Serafini M, Lagerros YT, Nyren O, La Vecchia C, Rossi M, Ye W, Tavani A, Adami H-O, Grotta A, Bellocco R, Dietary Antioxidant Capacity and Risk of Stroke in a prospective cohort study of Swedish Men and Women, Nutrition (2016), doi: 10.1016/j.nut.2016.07.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Dietary Antioxidant Capacity and Risk of Stroke in a prospective cohort study of
2
Swedish Men and Women
3
Luca Colarusso MSc*1, Mauro Serafini PhD*2, Ylva Trolle Lagerros MD PhD3,4, Olof
4
Nyren MD PhD5, Carlo La Vecchia MD6, Marta Rossi ScD7,8, Weimin Ye MD PhD5,
5
Alessandra Tavani ScD7, Hans-Olov Adami MD PhD5,8, Alessandra Grotta* PhD5,
6
Rino Bellocco* ScD5,9.
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1
RI PT
Department of Statistics and Quantitative Methods, University of Milano-Bicocca, Milan, Italy. 2
Functional food and metabolic stress prevention laboratory, Center for Food and Nutrition, Council for Agricultural Research and Economics CREA, Rome, Italy. 3
SC
Department of Medicine, Clinical Epidemiology Unit, Karolinska Institutet, Stockholm, Sweden. 4
M AN U
Department of Medicine, Clinic of Endocrinology, Metabolism and Diabetes, Karolinska University Hospital Huddinge, Stockhlom, Sweden. 5
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. 6
Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy. Department of Epidemiology, Mario Negri Institute, Milan, Italy.
8
Department of Epidemiology, Harvard School of Public Health, Boston MA, USA.
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9
*
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Department of Statistics and Quantitative Methods, University of Milano-Bicocca, Milan, Italy.
These authors contributed equally to the manuscript.
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Correspondence to: Rino Bellocco, Sc.D., Department of Statistics and Quantitative Methods, University of Milano-Bicocca, Milan, Italy.
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E-mail [email protected]; phone number: +39 02 64485831; fax number: +39
41
02 64485899
ACCEPTED MANUSCRIPT Abstract
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Objective: Both observational studies and randomized trials have shown that a diet
44
rich in antioxidants can reduce systemic inflammation and oxidative stress, two
45
conditions that, together with obesity and smoking, are established risk factors for
46
stroke. However, the association between antioxidant intake and risk of stroke is
47
poorly understood, particularly when studying possible interaction with gender. We
48
investigated the relationship of Non Enzymatic Antioxidant Capacity (NEAC) on risk
49
of stroke in a large Swedish prospective cohort.
50
Methods: This cohort study included 34,555 men and women from the Swedish
51
National March Cohort. NEAC was assessed using a detailed food frequency
52
questionnaire, collected at baseline. We achieved complete follow-up from enrolment
53
in 1997 through 2010 by record linkage to nation-wide registers.
54
We identified 1,186 incident cases of a first stroke, of which 860 were ischemic, 201
55
hemorrhagic and 125 unspecified. We used multivariable Cox proportional hazards
56
models to estimate adjusted hazard ratios (HR) with 95% confidence intervals (CI).
57
Results: Compared to women in the lowest quartile of NEAC women in the highest
58
quartile had a 27% lower incidence of total stroke (HR=0.73; 95% CI: 0.53-0.99; p
59
for trend = 0.03) and 35% lower incidence of ischemic stroke (HR=0.65; 95% CI:
60
0.43-0.99; p for trend =0.01). Among men, the relationship between NEAC and risk
61
of stroke was not statistically significant and all HRs were close to unity.
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Conclusion: Our findings suggest that dietary antioxidant capacity from different
SC
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Atherosclerosis; Risk Factors; Cohort Studies.
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foods and beverages is inversely associated with risk of stroke, more specifically ischemic stroke, in women. Keywords: Stroke; Antioxidant Effect; Dietary Habits; Epidemiology;
2
ACCEPTED MANUSCRIPT Introduction
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Stroke is the second leading cause of death in the world [1]. Well-known modifiable
69
risk factors for this neurovascular emergency include hypertension, obesity, physical
70
inactivity, an unhealthy diet and smoking [2]. Furthermore, oxidative and
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inflammatory stress promotes atherosclerosis [3] that ultimately can lead to stroke [2,
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4]. A high consumption of flavonoids and phytochemicals with antioxidant properties
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contained in fruits and vegetables is associated with reduced oxidative stress [5] and
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reduced systemic inflammation [6, 7]. Antioxidants in fruits, vegetables, and other
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foods and beverages may therefore prevent stroke and other cardiovascular diseases
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by reducing excessive production of free radicals induced by oxidative and
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inflammatory stress [8-15].
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However, few observational studies have investigated the association between total
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antioxidant capacity from diet and risk of stroke [10, 11, 16]. Two studies found an
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inverse association between consumption of antioxidants and risk of stroke. A third
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study did not find any evidence of neither a positive, nor a negative association [16].
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Therefore, the association remains open for discussion. Moreover, a possible
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interaction with gender has not been investigated yet. However, previous studies have
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suggested a possible gender difference in antioxidant status [17], which may in turn
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lead to a potential different effect of dietary antioxidants on the risk of stroke.
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capacity assays such as ferric reducing antioxidant potential (FRAP) [20].
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Non Enzymatic Antioxidant Capacity (NEAC), also known as total antioxidant capacity (TAC), is a measure proposed to assess the cumulative power offered by all antioxidant sources from diet [18, 19]. NEAC can be estimated through antioxidant
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We investigated the relationship of NEAC, estimated using food item-specific
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antioxidant FRAP values, on risk of stroke in a large Swedish prospective cohort of
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women and men.
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Methods
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The Swedish National March Cohort (SNMC) is a prospective study established in
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Sweden in 1997 during a four-day national fund-raising event promoted by the
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Swedish Cancer Society. Participants in the SNMC were invited to fill out a 36-page
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questionnaire concerning socio-demography, lifestyle, diet and medical history at
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baseline. An 85-item food frequency questionnaire (FFQ), a slightly abbreviated
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version of a validated 96-item questionnaire [21], was used to estimate individual
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intake of common Swedish food items. The participants were asked to indicate how
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often, on average in the previous year, they had consumed these foods and beverages.
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Eight response categories ranged from “never/seldom” to “3 or more times per day”.
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We computed body mass index (BMI) by dividing reported weight (kg) by the
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squared height (m) and categorized it according to the World Health Organization
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classification (underweight: <18.5; normal weight: 18.5 to < 25 kg/m2; overweight: 25
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to < 30 kg/m2; obese: ≥ 30 kg/m2). Physical activity was assessed by a validated
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questionnaire [22]; time spent on each of nine predefined intensity levels of physical
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activity during a typical weekday was self-estimated, and the estimates were
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multiplied by their respective metabolic equivalent (MET) values [22]. Finally, the products were summed up and total physical activity was reported in terms of METh/day.
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Food item-specific antioxidant capacity values were obtained from a database with
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“in vitro” measurements using the FRAP assay [20, 23]. A total of 66 out of the
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original 85 FFQ items had FRAP values in the database and were used for dietary
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NEAC computation. These values were multiplied by the reported frequency of
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consumption taking the portion size into account. Coffee consumption was left out
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when computing NEAC due to the observed discrepancy between the “in vivo” and
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“in vitro” effects of the Maillard products [24, 25]. Other foods, such as chocolate and
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whole grains, were not excluded in the antioxidant computation since they have a
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lower content of melanoidins. Moreover, these foods have a high level of other
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bioactive ingredients such as flavonoids. We have followed the same approach in a
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previous study [26]. Since FRAP values for dietary supplements were not available,
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antioxidant supplements were left out of NEAC computation.
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The reproducibility and validity of NEAC values derived from food frequency
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questionnaire data combined with FRAP assays are reported in the paper from
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Rautiainen et al. [21]; the correlation between NEAC was adjusted for total energy
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intake using the residual method [27], since estimated NEAC correlated with total
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energy intake (ρ=0.4; p <0.05). The validity of the dietary food items contributing to
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NEAC compared to food record was acceptably good in a similar Swedish cohort.
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The estimated Pearson correlation coefficients ranged from 0.4 to 0.8 (A. Wolk,
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unpublished data).
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Follow-up
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ACCEPTED MANUSCRIPT A total of 43,863 men and women completed the questionnaire and consented to
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follow-up through record linkages with multiple national registries using the
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individually unique national registration numbers to ensure perfect matching. We
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excluded from the analyses 1,740 participants under the age of 18, 19 participants
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with invalid national registration number or conflicting answers, 457 who were
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recorded as emigrated before the start of follow-up, and 4,045 with any recorded
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cardiovascular diagnosis (e.g., stroke, myocardial infarction, heart failure, angina
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pectoris, and atrial fibrillation; International Classification of Diseases, [ICD7] 330-
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334, 400-468, [ICD8] 390-458, [ICD9] 390–459, and [ICD10] I00-I99 before the start
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of follow-up. We also excluded 2,652 participants with a previous history of cancer
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recorded in the Swedish Cancer Register (except non-melanoma skin cancer), and 406
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participants with extreme values for total energy intake (± 3 standard deviations for
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the mean value of loge transformed energy, ≤927 or >5,311 kcal for men, and ≤870 or
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>4,348 kcal for women). The remaining 34,555 participants were followed from
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October 1, 1997 until time of first hospitalization for stroke, emigration, death, or
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December 31, 2010, whichever occurred first. Incident stroke cases were identified in
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the nationwide and essentially complete registers of Inpatient Care and Causes of
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Death using the following ICD10 codes: I63.0-I63.5, I63.8-I63.9 (cerebral infarction);
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I61 (intracerebral haemorrhage); I60 (subarachnoid haemorrhage); I64 (unspecified
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stroke).
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variable. Descriptive statistics were presented for the whole cohort according to sex-
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specific quartiles of NEAC intake. We used Cox proportional hazards regression
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Statistical Analysis
Dietary NEAC values were categorized in quartiles, but also analyzed as a continuous
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ACCEPTED MANUSCRIPT models to assess the association between dietary NEAC intake and stroke risk. Hazard
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ratios (HRs) and the corresponding 95% confidence intervals (CIs) were obtained
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using age as the underlying time scale; they were calculated for categorical NEAC
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sex-specific quartiles using the lowest quartile as the reference category. The
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multivariable models were adjusted for the following potential confounders: sex, BMI
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(<18.5, 18.5-24.9, 25.0-29.9, 30+ kg/m2), alcohol consumption (never, low: ≤1 times
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per week, medium: >1 to 6 times per week and, high: ≥7 times per week, based on the
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frequency of drinking), smoking status (never, former and current smokers), physical
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activity (METh/day), educational level (<12 years or ≥12 years), dietary supplement
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use (yes/no), self-reported hypertension (yes/no), self-reported diabetes (yes/no),
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coffee consumption (daily consumption in quartiles), aspirin use (yes/no), self-
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reported lipid disturbance (yes/no) and, only for women, hormone replacement
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therapy use (yes/no). Moreover, we repeated our analyses by adjusting for self-
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reported menopausal status. To assess the proportional hazards assumption, we used
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the deviance test based on scaled Schoenfeld residuals. The proportional hazards
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assumption was satisfied in almost all multivariable models; when this assumption
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was not fulfilled, stratified Cox regression models were fitted.
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We assessed a linear trend across quartiles of NEAC using the median value of each
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NEAC category and including the resulting series of values in the models as a
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continuous variable. The validity of the assumption of a linear relationship between
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subgroup analyses according to sex, age (≤60, >60 years), BMI (<25, ≥25 kg/m2),
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NEAC and the stroke incidence was evaluated in Cox regression by adding a quadratic term and testing its significance. We also used a restricted cubic spline with 3 knots corresponding to the 5th, 50th and 75th percentiles [28]. We performed
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hypertension (yes/no), diabetes (yes/no), smoking (yes/no) and dietary supplement
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use (yes/no). We tested for statistical significance of effect modification on a
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multiplicative scale. Deviation from multiplicativity was evaluated using the
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likelihood ratio (LRT) test to compare nested models.
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The proportion of missing data on covariates were the following: 11.9% for total
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physical activity, 7.7% for smoking status, 4.9% for self-reported diabetes, 4.9% for
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BMI, 4.2% for self-reported lipid disturbance, 3.2% for self-reported hypertension;
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for the other covariates the proportion of missing information was below 2%.
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We used multiple imputation techniques to assess the robustness of estimates based
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on complete data to the presence of missing values. Under the assumption of data
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missing at random, we fitted multiple imputation models based on chained equations
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[29, 30], generating five imputed datasets from the initial cohort. HRs from the
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imputed datasets were combined using Rubin’s rules producing the pool effect
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estimate and relative estimated standard errors.
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To investigate the possible impact of reverse causality on the results we excluded
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cases during the first 3 years of follow-up to account for changes in dietary habits due
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to undiagnosed cardiovascular disease. We also repeated our main analyses using
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calendar time as time scale and adjusting for age.
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All probability values were presented as two-sided and p-values < 0.05 were
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considered statistically significant. All statistical analyses were performed using Stata
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(Version 13.1; StataCorp LP). 8
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The Regional Ethics Review Board at the Karolinska Institutet approved the study.
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Results
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Table 1 shows baseline characteristics of the cohort according to sex-specific quartiles
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of NEAC. Median NEAC intake was slightly lower in men compared to women. The
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major contribution to NEAC came from fruit and vegetable consumption (27.6% in
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women, 22.5% in men), tea (29.0% in women, 24.0% in men), whole grains (8.7% in
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women, 11.3% in men) and chocolate (9.3% in women and 10.3% in men). Men and
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women in the highest quartile of dietary NEAC were on average older than those in
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the lowest quartile. They were also more likely to use dietary supplements, to
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consume more fruits and vegetables, less coffee, to be non-smokers, and to have a
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higher education.
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During 438, 272 person-years of follow-up 1,186 participants had a first stroke (657
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women, 529 men). Of these stroke events, 860 were ischemic, 201 hemorrhagic and
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125 unspecified. Table 2 and Table 3 report results from separate Cox regression
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models for each of the subtypes as well as total stroke, adjusted for age and potential
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confounders and stratified according to sex (p for interaction on the multiplicative
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scale between sex and NEAC for total stroke=0.02). Among women, an inverse
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association between NEAC intake and total stroke incidence was found. Females in the highest quartile of NEAC had a 27% (95% CI: 0.53-0.99) lower incidence of total
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stroke, compared with those in the lowest quartile with a statistically significant linear
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trend in risk (p for trend=0.03). The observed inverse association with NEAC intake 9
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in women was driven mainly by the trend observed for ischemic stroke. Women with
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the highest NEAC intake had a 35% lower incidence of ischemic stroke (95% CI:
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0.43-0.99; p for trend=0.01) while no clear associations were found with hemorrhagic
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stroke or unspecified stroke, admittedly based on small numbers. After adjusting for
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menopausal status, results did not change substantially. Among men, we were unable
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to confirm any association between dietary NEAC and stroke of any type (Table 3).
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The association between NEAC and risk of stroke in women did not change
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significantly by age, history of diabetes, hypertension, alcohol consumption, and
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dietary supplement use.
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When re-running the same statistical analyses to assess the potential impact of the
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missingness on the observed associations, HRs based on multiple imputed datasets
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confirmed findings from complete data analysis (data not shown). In addition, we
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observed similar patterns when the exposure was treated as a continuous variable.
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Finally, when we excluded cases occurring in the first 3 years of follow-up, results did
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not change materially: women in the highest, as compared with the lowest quartile of
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NEAC intake had a 27% and 34% lower hazard of total stroke (HR=0.73; 95% CI:
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0.53-1.00; p for trend: 0.05) and ischemic stroke (HR=0.66; 95% CI: 0.39-0.98; p for
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trend=0.04), respectively. We observed the same results when using calendar time as
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time scale and adjusting for age at recruitment.
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Discussion
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In this large prospective study, dietary NEAC intake was associated with a reduced
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incidence of stroke, more specifically ischemic stroke, in women. Compared with
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women in the lowest quartile of dietary NEAC, those in the highest quartile had a
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35% lower incidence of ischemic stroke, and a 27% lower incidence of total stroke.
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No consistent association was found in men.
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Epidemiological studies have found an inverse association between risk of stroke and
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single food items such as tea [31], chocolate [32, 33], fruits and vegetables [8] and
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cereals [34-36]. Further, randomized intervention studies [37, 38] have highlighted
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the role of antioxidants from diet in preventing or mitigating systemic inflammation,
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oxidative stress and atherosclerosis.
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Therefore, the concept that dietary antioxidant capacity may play a role in stroke
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incidence is not completely new. However, to our knowledge, only three previous
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studies [10, 11, 16] investigated the association between the overall antioxidant
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capacity, rather than individual contributions from single food items, and the risk of
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stroke.
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Our results are consistent with two of these studies showing a marginally significant
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inverse association between NEAC and the risk of stroke [10, 11]. The first one, the
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that a high NEAC intake was inversely associated with total stroke among women
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Italian section of the European Prospective Investigation into Cancer and Nutrition (EPIC), found that dietary NEAC was inversely related to ischemic stroke, but not with hemorrhagic stroke. The second one, the Swedish Mammography Cohort, found
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quartile of NEAC (HR=0.83; 95% CI: 0.70-0.95). Our data suggest a role of NEAC in
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preventing stroke in women. A greater role of dietary NEAC on the relative risk of
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stroke in women compared to men is conceivable, considering the difference in
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background incidence, and the higher levels of NEAC in women’s diet. An unclear
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increase in the incidence of ischemic stroke is found among men with the highest
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NEAC intake. This result is not straightforward to interpret and indicates the need for
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further studies investigating different effects of NEAC by gender.
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The stronger association found with ischemic rather than hemorrhagic stroke is not
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surprising, since diet and lifestyle have been shown to be stronger associated with
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ischemic than hemorrhagic stroke [15, 39, 40]. The reduction in stroke incidence
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observed among women with a high dietary antioxidant intake can be related to the
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antioxidants’ ability to counteract the circulating levels of free radicals that are
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involved in the systemic inflammation and oxidative stress. Another important aspect
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to take into account involves the homeostatic redox process and its balancing
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mechanism between endogenous and exogenous antioxidants. When oxidative stress
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is absent, endogenous defenses are sufficient to hinder the production of free radicals.
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But when oxidative stress is ongoing, endogenous antioxidants are not able to inhibit
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the production of free radicals efficiently. Therefore, the contribution of exogenous
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antioxidants from diet may be crucial to support the endogenous redox machinery.
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A third study, the Rotterdam Study [16] did not find an association between NEAC intake and risk of stroke. This may depend on several factors, including a smaller
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sample size and different exclusion criteria, other major sources of NEAC, and a
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higher mean age.
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The fact that the consumption of the most important dietary sources of antioxidants in
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our study (fruits and vegetables, and tea) was lower among men than among women
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reduces our power to detect an association. When considering the 19 food items not
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included in the NEAC computation, there were no particular differences between men
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and women in the food frequency consumption that could somewhat explain the
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observed association among women, but not among men.
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NEAC computation originates from the hypothesis that antioxidants from foods may
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work as antioxidants in vivo, which is an extremely complex aspect of the dietary
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modulation of oxidative stress and disease prevention; measurements of biological
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biomarkers would be needed in order to better understand the mechanism. However,
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we believe that in vitro NEAC estimates can be useful to guide researchers to better
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understand the antioxidant effect on humans, but further studies on plasma NEAC
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could help to understand the real mechanism of antioxidant action.
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Our study has several strengths. The most important one is the use of NEAC as a
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measure of the overall antioxidant potential from the diet. Other important strengths
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are the prospective design, the detailed data on diet and potential risk factors and the
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large sample size. Furthermore the large number of stroke cases and record linkage to
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nation-wide registers allowed us for a nearly complete follow-up of study outcomes.
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information on bioavailability. Foods with the highest content of antioxidants may not
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The most important limitation is due to the fact that, although dietary assessment of NEAC is correlated with plasma antioxidant levels [21], it does not provide direct
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vivo. In fact, the use of a coefficient of absorption based on previous evidences from
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ingestion studies, has recently been proposed in the literature [12], but this approach
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could be questionable. Another limitation is the use of only one semi-quantitative
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food frequency questionnaire which allow assessment of the frequencies of
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consumption, but not the exact amount of food consumption, and it does not consider
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the use of antioxidant supplements. Finally, the use of FRAP values calculated from a
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non-Swedish food database items [20, 23]. It should be noticed that geographic
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location and growing conditions might not entirely explain the observed within study
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variability and therefore, using antioxidant values from countries with eating habits
348
similar to those in Sweden may not be more appropriate than using an Italian food
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items database.
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Another limitation could be represented by the fact that FRAP values for dietary
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supplements were not available and our results cannot be related to antioxidant
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supplements, but only to food items.
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Conclusion
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In conclusion, this study shows that a high-NEAC diet is associated with a reduced
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risk of ischemic (and total) stroke in women, and suggests a potential role of a diet
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rich in fruit, vegetables and, food with antioxidant properties in the prevention of the
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disease.
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Acknowledgments
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This work was conducted with the contribution of the Italian Ministry of University
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and Research (PRIN 2009 X8YCBN), the Swedish Cancer Society (Grant CAN 14
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(Dnr: 2368/10-221); and the regional agreement on medical training and clinical
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research between Stockholm County Council and Karolinska Institutet for Ylva Trolle
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Lagerros. The authors would like to thank Keith Humpreys (Associate Professor of
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Biostatistics, Department of Medical Epidemiology and Biostatistics, Karolinska
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Institutet) for language editing.
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Conflict of Interest: none.
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[32] Buijsse B, Weikert C, Drogan D, Bergmann M, Boeing H. Chocolate consumption in relation to blood pressure and risk of cardiovascular disease in German adults. European Heart Journal. 2010;31:1616-23. [33] Mink PJ, Scrafford CG, Barraj LM, Harnack L, Hong C-P, Nettleton JA, et al. Flavonoid intake and cardiovascular disease mortality: a prospective study in postmenopausal women. American Journal of Clinical Nutrition. 2007;85:895-909. [34] Ascherio A, Rimm EB, Hernan MA, Giovannucci EL, Kawachi I, Stampfer MJ, et al. Intake of potassium, magnesium, calcium, and fiber and risk of stroke among US men. Circulation. 1998;98:1198-204. [35] Larsson SC, Männistö S, Virtanen MJ, Kontto J, Albanes D, Virtamo J. Dietary fiber and fiber-rich food intake in relation to risk of stroke in male smokers. European Journal of Clinical Nutrition 2009;63:1016-24. [36] Oh K, Hu FB, Cho E, Rexrode KM, Stampfer MJ, Manson JE, et al. Carbohydrate intake, glycemic index, glycemic load, and dietary fiber in relation to risk of stroke in women. American Journal of Epidemiology. 2005;161:161-9. [37] Brighenti F, Valtuena S, Pellegrini N, Ardigo D, Del Rio D, Salvatore S, et al. Total antioxidant capacity of the diet is inversely and independently related to plasma concentration of high-sensitivity C-reactive protein in adult Italian subjects. British Journal of Nutrition. 2005;93:619-25. [38] Valtueña S, Pellegrini N, Franzini L, Bianchi MA, Ardigò D, Del Rio D, et al. Food selection based on total antioxidant capacity can modify antioxidant intake, systemic inflammation, and liver function without altering markers of oxidative stress. American Journal of Clinical Nutrition. 2008;87:1290-7. [39] Misirli G, Benetou V, Lagiou P, Bamia C, Trichopoulos D, Trichopoulou A. Relation of the traditional mediterranean diet to cerebrovascular disease in a mediterranean population. American Journal of Epidemiology. 2012;176:1185-92. [40] Miglio C, Peluso I, Raguzzini A, Villaño DV, Cesqui E, Catasta G, et al. Fruit juice drinks prevent endogenous antioxidant response to high-fat meal ingestion. British Journal of Nutrition. 2013:1-7.
M AN U
452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481
AC C
EP
TE
D
482
17
ACCEPTED MANUSCRIPT 483 484
Table 1: Age-standardized baseline characteristics of participants in the Swedish
485
National March Cohort: A) women B) men
Women (n=22,712)
RI PT
Non-Enzymatic Antioxidant Capacity* Q1 (n=5,678)
Q2 (n=5,678)
Q3 (n=5,678)
Q4 (n=5,678)
5.8 (5.1-6.6)
8.3 (7.8-8.9)
10.8 (10.1-11.6)
15.2 (13.6-17.9)
Age, years, mean(SD)
48.0 (15.0)
48.9 (15.2)
48.8 (15.2)
49.7 (15.0)
>12 years of education,
36.0
42.3
46.9
55.8
8.5
7.6
6.1
24.6 (3.8)
24.3 (3.7)
24.1 (3.5)
23.9 (3.5)
TE
(A)
37.5 (11.6)
37.7 (11.6)
37.7 (11.9)
Median NEAC (IQR)
SC
(mmol/d)
% 11.6
D
Current smokers, % Body Mass Index, mean kg/m2(SD)
37.2 (11.9)
EP
Total Physical Activity
M AN U
Baseline characteristics
(SD)
AC C
Score (MET*h/d), mean
Aspirin use, %
65.4
66.2
65.9
64.5
Dietary supplement use,
50.4
53.1
55.1
57.0
%
18
ACCEPTED MANUSCRIPT Self-reported
10.7
10.4
9.9
11.0
Self-reported diabetes, %
1.7
1.7
1.8
2.0
Self-reported lipid
2.8
2.3
2.6
2.2
0.7
1.0
1.1
1.7
24.9
25.8
1948.8 (496.4)
2048.0 (496.3)
hypertension, %
Alcohol consumption
RI PT
disturbance, %
(high), % Hormone replacement
27.0
Total energy intake
SC
use %
3.6 (1.7)
4.6 (2.0)
5.2 (2.4)
5.6 (2.9)
1.2 (0.4)
1.3 (0.5)
1.4 (0.5)
1.4 (0.5)
1.3 (0.7)
1.8 (0.9)
3.1 (2.0)
Foods, mean (SD) Fruits and vegetables, servings/d
D
Whole Grains,
TE
servings/d
1.0 (0.2)
EP
Tea, servings/d
1961.1 (511.7)
M AN U
(kcal/d), mean (SD)
2065.1 (500.0)
29.0
0.8 (0.7)
1.1 (1.1)
1.3 (1.5)
1.4 (2.0)
Coffee, servings/d
3.3 (1.6)
3.0 (1.5)
2.6 (1.4)
2.0 (1.6)
25.6 (8.7)
25.9 (8.5)
25.4 (8.4)
23.5 (8.5)
AC C
Chocolate, servings/wk
Nutrients, mean(SD) Saturated fatty acids, gr/day
19
ACCEPTED MANUSCRIPT Monounsaturated fatty
18.6 (5.8)
19.1 (5.9)
19.0 (5.9)
17.5 (5.8)
6.0 (2.0)
6.5 (2.0)
6.6 (2.1)
6.3 (2.1)
acids, gram/day Polyunsaturated fatty acids, gram/day
Non-Enzymatic Antioxidant Capacity*
RI PT
(B) Men (n=11,843)
Q1 (n=2,961)
Q2 (n=2,961)
Q3 (n=2,961)
Q4 (n=2,960)
Median NEAC (IQR)
5.4 (4.7-5.9)
7.4 (6.9-7.9)
9.4 (8.9-10.1)
13.2 (11.8-15.2)
SC
(mmol/d)
M AN U
Baseline characteristics Age, years, mean(SD)
46.8 (17.9)
49.5 (17.5)
51.7(16.7)
54.4 (15.1)
>12 years of education,
36.7
41.4
47.6
55.0
7.8
5.6
4.9
25.1 (3.2)
24.9 (3.3)
24.6 (3.2)
44.1 (17.5)
44.0 (15.9)
42.9 (15.3)
42.3 (15.4)
Aspirin use, %
60.8
60.9
61.3
58.9
Dietary supplement use,
30.7
35.5
40.0
43.0
%
EP
kg/m2(SD)
25.4 (3.3)
TE
Body Mass Index, mean
10.5
D
Current smokers, %
Total Physical Activity
(SD)
AC C
Score (MET*h/d), mean
20
ACCEPTED MANUSCRIPT % Self-reported
9.1
9.1
9.5
9.3
Self-reported diabetes, %
3.0
2.6
2.3
2.3
Self-reported lipid
2.2
2.3
2.6
2.5
2.6
2.2
2227.8 (616.0)
2344.5 (585.6)
hypertension, %
Alcohol consumption (high), %
(kcal/d), mean (SD)
Fruits and vegetables,
2.7 (1.4)
2358.3 (618.3)
2269.9 (653.9)
3.5 (1.7)
4.0 (2.0)
4.3 (2.3)
1.5 (0.5)
1.6 (0.5)
1.6 (0.6)
1.0 (0.2)
1.3 (0.5)
1.8 (0.7)
3.1 (1.5)
0.7 (0.7)
1.1 (1.1)
1.4 (1.5)
1.7 (2.2)
4.7 (1.8)
4.4 (1.8)
4.0 (1.8)
3.5 (2.0)
29.5 (10.7)
29.5 (9.8)
29.0 (10.3)
27.4 (10.6)
21.0 (6.9)
21.7 (6.6)
21.5 (6.9)
20.5 (7.3)
servings/d Whole Grains,
1.4 (0.5)
EP
TE
D
servings/d
Chocolate, servings/wk
4.7
M AN U
Foods, mean (SD)
Tea, servings/d
3.5
SC
Total energy intake
RI PT
disturbance, %
Coffee, servings/d
AC C
Nutrients, mean(SD) Saturated fatty acids, gr/day
Monounsaturated fatty
21
ACCEPTED MANUSCRIPT acids, gram/day Polyunsaturated fatty
6.6 (2.3)
7.2 (2.3)
7.3 (2.5)
7.1 (2.6)
acids, gram/day *NEAC was estimated through ferric reducing antioxidant power (FRAP); Q, quartile; MET, metabolic equivalents.
AC C
EP
TE
D
M AN U
SC
RI PT
486
22
ACCEPTED MANUSCRIPT 487 488
Table 2: Dietary Non-enzymatic antioxidant capacity (NEAC) and risk of
489
stroke in the Swedish National March Cohort among women.
WOMEN
Non-Enzymatic Antioxidant Capacity* Q1
Q2
Q3
Q4
p for
Total Stroke
RI PT
trend
168
182
163
Person-years,
72,876
72,650
72,794
Age-adjusted HR
1.0
0.99 (0.81-1.22)
0.89 (0.72-1.11)
Multivariable HR†
1.0
0.99 (0.75-1.31)
No. of cases
116
Age-adjusted HR Multivariable HR†
0.93 (0.69-1.24)
0.73 (0.53-0.99)
0.03
144
108
99
1.0
1.13 (0.89-1.44)
0.85 (0.66-1.11)
0.73 (0.56-0.95)
<0.01
1.0
1.20 (0.85-1.68)
D
0.93 (0.65-1.34)
0.65 (0.43-0.99)
0.01
M AN U
<0.01
TE EP
No. of cases
72,652
0.74 (0.59-0.92)
Ischemic
Haemorrhagic
144
SC
No. of cases
34
24
38
29
1.0
0.67 (0.40-1.13)
1.01 (0.67-1.68)
0.77 (0.47-1.27)
0.61
1.0
0.58 (0.30-1.21)
1.1 (0.62-1.95)
0.85 (0.45-1.60)
0.97
No. of cases
18
14
17
16
Age-adjusted HR
1.0
0.71 (0.35-1.42)
0.86 (0.44-1.67)
0.74 (0.38-1.46)
0.53
Multivariable HR†
1.0
0.74 (0.32-1.72)
0.49 (0.19-1.29)
0.84 (0.35-2.02)
0.71
AC C
Age-adjusted HR
Multivariable HR† Unspecified
23
ACCEPTED MANUSCRIPT 490
* NEAC was estimated through ferric reducing antioxidant power (FRAP); Q, quartile (Q1
491
reference).
492
† Adjusted for age, education level, smoking status, body mass index, physical activity, self-
493
reported hypertension, self-reported diabetes, aspirin use, dietary supplement use, coffee
494
consumption, alcohol consumption, self-reported lipid disturbance, and total energy intake.
495
AC C
EP
TE
D
M AN U
SC
RI PT
496
24
ACCEPTED MANUSCRIPT 497
498
Table 3: Dietary Non Enzymatic Antioxidant Capacity (NEAC) and risk of stroke in the Swedish
499
National March Cohort among men.
Non-Enzymatic Antioxidant Capacity* Q1
RI PT
MEN
Q2
Q3
106
Person-years,
37,116
Age-adjusted HR
1.0
Multivariable HR†
1.0
trend
168
36,942
36,779
36,585
1.0 (0.77-1.29)
0.97 (0.75-1.25)
1.10 (0.87-1.41)
0.36
1.19 (0.85-1.67)
0.99 (0.70-1.40)
1.35 (0.96-1.89)
0.11
93
98
129
1.0
1.08 (0.80-1.47)
1.04 (0.77-1.41)
1.22 (0.92-1.63)
0.17
1.0
1.27 (0.86-1.88)
1.08 (0.73-1.61)
1.55 (1.07-2.33)
0.05
No. of cases
18
18
16
24
Age-adjusted HR
1.0
0.88 (0.46-1.70)
0.72 (0.37-1.41)
0.97 (0.53-1.79)
0.99
Multivariable HR†
1.0
1.0 (0.41-2.51)
0.74 (0.29-1.89)
0.91 (0.37-2.25)
0.81
EP
Age-adjusted HR
TE
73
AC C
No. of cases
p for
131
D
Ischemic
124
M AN U
No. of cases
SC
Total Stroke
Q4
Multivariable HR† Haemorrhagic
25
ACCEPTED MANUSCRIPT Unspecified No. of cases
15
13
17
15
Age-adjusted HR
1.0
0.73 (0.35-1.54)
0.88 (0.44-1.77)
0.69 (0.34-1.42)
0.43
Multivariable HR††
1.0
0.80 (0.29-2.24)
0.68 (0.24-1.95)
0.56 (0.18-1.76)
0.34
* NEAC was estimated through ferric reducing antioxidant power (FRAP); Q, quartile (Q1
501
reference).
502
†Adjusted for age, education level, smoking status, body mass index, physical activity, self-
503
reported hypertension, self-reported diabetes, aspirin use, dietary supplement use, coffee
504
consumption, alcohol consumption, self-reported lipid disturbance, and total energy intake.
505
†† Adjusted for age, education level, smoking status, body mass index, physical activity, self-
506
reported hypertension, self-reported diabetes, dietary supplement use, coffee consumption,
507
alcohol consumption, self-reported lipid disturbance, and total energy intake and stratified by
508
aspirin use because the proportionality assumption did not hold.
M AN U
SC
RI PT
500
509 510
514 515 516 517 518
TE EP
513
AC C
512
D
511
26
ACCEPTED MANUSCRIPT HIGHLIGHTS: Dietary NEAC is a measure of the overall antioxidant capacity of diet. Dietary NEAC takes into account synergistic interactions among dietary antioxidants. The relationship between dietary NEAC and risk of stroke has been not extensively explored. High levels of dietary NEAC are associated with a decreased risk of ischemic stroke among women.
AC C
EP
TE
D
M AN U
SC
RI PT
1. 2. 3. 4.