PRINCIPAL INVESTIGATORS: Tomi-Pekka Tuomainen, MD, PhD, Virtanen Jyrki, PhD, Sari Voutilainen, PhD

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1 PRINCIPAL INVESTIGATORS: Tomi-Pekka Tuomainen, MD,, Virtanen Jyrki,, Sari Voutilainen, Project title: Finnish Vitamin D Trial FIND (Clinicaltrials.gov: NCT ) Site of research: University of Eastern Finland, Kuopio Date of research plan: Sept 15, 2015 I BACKGROUND Vitamin D insufficiency is widespread throughout the world. 1 There is abundant evidence from population studies that vitamin D deficiency is associated with higher risk of chronic diseases and other adverse health outcomes, but so far there is no data from large randomized clinical trials (RCT) in general population whether vitamin D status improvement by supplementation can improve health. Also the role of the inter-individual variation in the response to vitamin D supplementation, which appears to be quite marked, in determining the health effects of vitamin D need to be investigated. The classical role of vitamin D is in maintaining bone health, but in addition there is fairly consistent data from observational population studies on the benefits of higher serum 25(OH)D levels in the prevention of chronic diseases, such as cardiovascular disease (CVD), cancer, diabetes, hypertension, autoimmune diseases and infectious diseases. 2-5 In contrast, the results from RCTs have not been as promising. 6,7 However, most RCTs were not designed to evaluate the impact of vitamin D on non-skeletal outcomes, were small in size, or the dose of vitamin D has been too low or the duration too short, to be able to show an effect. Besides the clinically diagnosed health outcomes, vitamin D may also have an effect on how people perceive their own health, i.e. self-rated health. For eample, vitamin D deficiency has been associated with chronic pain and its comorbidities such as poor sleep quality, aniety, mood disorders and depression, 8 which can have a profound effect on well-being and quality of life. There is a biological basis to assume that vitamin D plays a role in pain pathophysiology, the maintenance of chronic pain and comorbidities. 8 Although some benefit has been observed in small RCTs, there is no data from large RCTs whether correction of vitamin D deficiency by supplementation can improve self-rated health outcomes. 8 Also, there is no data on whether vitamin D supplementation can improve perceived health in vitamin D sufficient individuals. Vitamin D, obtained from diet or produced in the skin, is hydrolyzed in the liver to calcidiol [25- hydroyvitamin D, 25(OH)D], a generally used indicator of vitamin D status in the body. In the kidneys 25(OH)D is further metabolized to the physiologically active form calcitriol [1,25(OH) 2 D], which is the high-affinity ligand of the transcription factor vitamin D receptor (VDR). 9 Besides the kidneys, 1,25(OH) 2 D is also synthesized locally in many tissues and cell types that epress VDR. Genome-wide more than 10,000 VDR binding sites have been found, and there are significant changes in the transcriptome profile in at least 1,000 genes. 10 There is significant inter-individual variability both in the vitamin D status and in the vitamin D response, 11 and it is very probable that a one-size-fits-all approach is not applicable for optimal vitamin D intake. Besides the environmental and demographic factors (diet, sunlight, season, latitude, race), several factors affect serum 25(OH)D concentrations or response to supplementation. These include, for eample, age, adiposity, gene polymorphisms, inflammation, medications, nutrient-nutrient interactions, and starting levels of 25(OH)D Furthermore, in the circulation about 90% of 25(OH)D is carried tightly bound to vitamin D binding protein (DBP), <10% to albumin and only <1% is in the free form. 17 However, it is postulated that the 25(OH)D that is not bound to DBP would represents the bioavailable 25(OH)D and would be a better marker for vitamin D status. 17 In other words, someone with low serum 25(OH)D with the current standard (<50 nmol/l) could have adequate levels of bioavailable 25(OH)D if the DBP levels are low or if there are certain genetic polymorphism in the gene 1

2 encoding DBP, which results in a low affinity to 25(OH)D. 17 Knowing the impact of vitamin D supplementation on the bioavailable vs. DBP-bound 25(OH)D would be etremely important in interpreting the results from vitamin D supplementation trials. II & III OBJECTIVES AND HYPOTHESES Objective 1: To investigate the impact of 5-year moderate-dose and high-dose vitamin D 3 supplementation on physiological effects and health outcomes in the ageing general population. Hypotheses Vitamin D supplementation has a dose-dependent, beneficial impact on both self-rated health (e.g pain, coherence, functional capacity) and physician-diagnosed illness (e.g cardiovascular disease, type 2 diabetes, cancer). Objective 2: To evaluate and identify genetic and environmental factors that contribute to the interindividual variation both in the response of serum 25(OH)D concentrations to supplementation and in health and health-related outcomes. Hypotheses There is large inter-individual variation in vitamin D responsiveness which is due to differences in i) the genetic makeup of individuals, ii) physiological processes not solely genetically determined (such as inflammation), and iii) dietary and other behaviorallyoriented environmental factors (e.g, medication and personality-related features, such as life management). The effects of vitamin D supplementation are dependent, at least to some etent, on the serum bioavailable non-dbp bound 25(OH)D. The effects of vitamin D supplementation are dependent, at least to some etent, on the individual responsiveness to changes in vitamin D metabolite concentrations. Objective 3: To investigate the impact of 5-year moderate and high-dose vitamin D supplementation on fractures, bone mineral density and body composition in the ageing general population. Hypotheses Vitamin D supplementation decreases the incidence of fractures and age-related regression in bone mineral density and affects beneficially body composition (primarily fat distribution) in a dose-dependent manner. IV RESEARCH METHODS Study design FIND started in September It is a 5-y trial of the benefits (and possible risks) of vitamin D supplementation on the incidence of major chronic diseases and on their risk factors among men 60 y and women 65 y from the general population who were free of CVD and cancer at baseline. Although several large RCTs have started, FIND is the only trial in such northern latitudes and one of the few RCTs with two different vitamin D doses. This provides a unique opportunity to investigate the dose-response of vitamin D supplementation on health and health-related events and states. The 2495 participants that started the study were randomized into three groups: 1) 40 µg of vitamin D 3, 2) 80 µg of vitamin D 3, or 3) placebo. A representative subgroup of 551 subjects were invited to visit the UEF Kuopio campus for more detailed investigations (Tables 1&2). Compliance, use of non-study drugs or supplements, diet, development of endpoints, and cancer and CVD risk factors are assessed by yearly questionnaires. Data on disease outcomes and on use of prescription medicines is also obtained by record linkage to the national registries. After the trial, the follow-up for major chronic disease events as well as for mortality will continue from for a minimum of 10 y. 2

3 The northern location and the use of two different vitamin D doses in the FIND study provide a significant and unique contribution to the overall knowledge of the health effects of vitamin D. The project will produce very valuable data to investigate the impact of long-term high-dose vitamin D supplementation on various risk factors, profiles, health-related characteristics and health outcomes. Outcomes ascertainment Morbidity and mortality Data on morbidity, mortality and medications is obtained from the national health registries. National Death Registry in collaboration with the Statistics Finland is used for the mortality data and the Hospital Discharge Registry (HILMO) governed by National Institute for Health and Welfare for data on acute health events. Information on medicine use is obtained from the nationwide Drug Prescription Registry at the Social Insurance Institution. Together with the medicine use data from the study forms, this information allows, among other things, to investigate whether vitamin D supplementation can reduce the number of drugs used. Self-rated health The annual study form includes sections with detailed questions that are aimed at assessing also the psychological and psychosomatic aspects of health, such as coherence, sleep, mood, mental health, functional capacity and pain. The pain-related questionnaires enable the detection of various pain conditions, like regional musculoskeletal pain, wide-spread pain and multi-site pain (pages 21-22). The most important pain features, i.e. the frequency, duration, intensity and interference of pain at the baseline and at the follow-up are recorded. In addition, many factors often associated with pain, such as self-rated health, functional capacity and earlier diagnosed somatic or psychiatric diseases are included in the analysis. Therefore, it is also possible to evaluate the interactions of depressive symptoms, comorbidities and functional capacity in the relationship between pain and vitamin D. Mood is evaluated with a brief version of Beck Depression Inventory, one of the most established mood questionnaires (page 26-27). This questionnaire evaluates the presence and severity of several depressive symptoms, such as subjective eperience of mood and interest in everyday activities. In addition to the medical and drug prescription records, this provides important information for investigation of the effects of vitamin D on self-rated health and provides opportunities for collaboration with eperts in these areas of research. Bone and body composition measurements Whole body dual-energy X-ray absorptiometry (DXA) measurement and bone ultrasound measurements are performed at baseline and at the trial s end at the Kuopio Musculoskeletal Research Unit of the UEF (professor H. Kröger). DXA and ultrasound can be used to investigate the impact of vitamin D supplementation on bone mineral density and DXA also on body composition (e.g. changes in fat mass and fat free mass in the whole body and in the different parts of the body, such as trunk and limbs). Eposures assessments Vitamin D metabolite analyses Effect of vitamin D supplementation on vitamin D status is obtained by determining the serum 25(OH)D concentrations. We will also measure the DBP-bound and unbound 25(OH)D, the active form 1,25(OH) 2 D, the inactive metabolite 24,25(OH) 2 D, and the vitamin D binding protein to investigate the effect of supplementation on vitamin D metabolism and their relation to health outcomes. Genetic analyses Vitamin D metabolism pathway involves many different players, some of which are coded genetically. Especially variation in the vitamin D receptor (VDR), GC (DBP), CYP2R1, and DHCR7 genes have been found to be influential These factors are strong determinants for vitamin D status, and therefore crucial to take into account when health effects of vitamin D supplementation are studied. The genes are to be genotyped at the Eastern Finland Genome Center (EFGC), UEF, Kuopio, and at the laboratory of Dr. Hyppönen in the University of South Australia. The EFGC is a well-equipped service core and has epertise to carry out the proposed analyses. Dr. Hyppönen, a known epert in vitamin D genetic variation and Mendelian randomization studies, is taking part in the design of the analyses. 3

4 Assessment of dietary and supplemental intakes Participants complete a validated 142-item food-frequency questionnaire (FFQ) at baseline, at 3 years and at trial s end. This will allow us to evaluate whether the effects of vitamin D supplementation vary by nutrient intakes. Collection of the dietary data will also give us a very good database of the modern dietary intake habits of the older population in Finland and also allows us to reliably eamine dietary changes over time. The FFQ can be filled either as a paper or an online version. Trained nutritionists check the FFQs. Power computations The study consists of 2495 subjects who are mainly followed by the annual study forms and national registry data. Among those, a subgroup of 551, about 183 in each study arm, were invited for more detailed investigations. The anthropometric and other on-site measurements and the genetic and biochemical measurements allow many continuous-type variable data analyses in the subgroup. Of these variables, e.g. circulating cytokines and glucose homeostasis parameters are of interest. For instance, for low dispersion variables (SD about 10% of the mean), like plasma glucose (mean 6.2 mmol/l, SD 0.4 in the recent VitDmet study, which included participants aged >60 y 22 ), the study is well powered (effect size of 5% requires N=30 per group with 1- =0.8 and =0.05). For medium dispersion variables (SD about half of the mean), like for plasma insulin (mean 11.8 mu/l, SD 5.9 in VitDmet), the study starts to be at its limits (effect size of 15% requires N=177 per group with 1- =0.8 and =0.05). For high dispersion variables (SD about the same as the mean), such as IL-6 (mean 1.66 pg/ml, SD 1.42 in VitDmet), the study is powered to detect differences with a large effect size of 25% (requiring 188 subjects per group), but slightly underpowered for a 20% effect size (requiring 289 subjects per group). Event-type data are analyzed in the entire set of the 2495 subjects. With this study size, >800 subjects per group, in pair-wise comparisons there needs to be a large effect size, about 25%, to reach a power of 80% with =0.05. This limitation will be overcome by conducting pooled and meta-analyses with other large interventional vitamin D supplementation studies, to increase power (see the section Study progress below). Ethical considerations and data management The study follows the principles of the Helsinki Declaration. All subjects are voluntary adults that have agreed to participate in the study. They are entitled to withdraw from the study at any time without eplanation for doing so. All subjects have signed a written informed consent. The appropriate approvals for the FIND have been obtained from the Ethics committee of the Kuopio University Hospital (decision #30/2010) and from the National Institute for Health and Welfare that administers the health registries (decision #THL/1608/ /2014). FIND is also registered at the Clinical Trials register (NCT ). Participants are monitored to possibilities and subjects with severe adverse effects (e.g. hypercalcemia) are immediately withdrawn from the study. Generally vitamin D is considered tolerable and safe in much higher intakes than what is used in the FIND study. In RCTs lasting up to one year, little evidence for toicity risk has been observed with doses up to 250 µg/d. 23 The Institute has a centralized research data storing and management system. The centralized data storing system enables the linkage to the national registries, and monitoring of the entries and any changes. Measures of confidentiality and data security are strictly enforced according to the UEF guidelines, registry policies, and national legislation. All study subjects are given a code number, which keeps their identity anonymous in any statistical analyses. These codes can be disclosed only by a data manager, who is responsible for the data records. All study data will be stored at the study centre, which has over 20 y of eperience in handling large population-based studies. 4

5 V RESEARCH TEAM AND ENVIRONMENT Research team The investigators, their merits and tasks are presented in the Table 3. Table 3. Investigators, merits and tasks Core investigator group Tomi-Pekka Tuomainen, MD,, Sari Voutilainen,, adjunct professor Jyrki Virtanen,, adjunct professor Tarja Nurmi, Jaakko Mursu,, adjunct professor Merits Co-PI, PI of the VitDmet trial. Professor of epidemiology. He has received an Academy of Finland Research Grant for the years and Co-PI. Nutritional epidemiologist and adjunct professor of public health. She has received an Academy of Finland Research Grant for the FIND study for the years and a 2-y post-doctoral researcher grant for the years Co-PI. Adjunct professor of nutritional epidemiology. Postdoctoral research fellow at the Harvard School of Public Health in He has received a 3-y post-doctoral researcher grant from the Academy of Finland for the years Chief biochemist. She has previously received a 3-y postdoctoral researcher grant from the Academy of Finland for the years Post-doctoral research fellow at the University of Minneapolis in He has been the PI of 7 smaller supplementation studies at the Institute. DNA & RNA analyses Analyses of vitamin D metabolites Data analyses Scientific publication Etended study group (The role of the investigators is to provide knowledge in their respective areas of epertise related to the project.) Antti Aro, MD, Emeritus professor, nutritional epidemiologist, National Institute for Health and Welfare. Jussi Kauhanen, MD, Professor, Head of the Institute of Public Health and Clinical Nutrition, UEF, Kuopio. Epertise in Public health, epidemiology, psychosomatic medicine. Heikki Kröger, MD, Professor of Orthopaedics and Traumatology, Kuopio Musculoskeletal Research Unit. PI of the Kuopio Osteoporosis Risk Factor and Prevention Study (OSTPRE). Christel Professor, Head of Department of Applied Chemistry and Microbiology, University Lamberg-Allardt, of Helsinki. Long epertise in vitamin D research. E.g. invited chair of groups for vitamin D as well as calcium for the Nordic Nutrition Recommendations Soili Lehto, MD, Adjunct professor in eperimental psychiatry, Department of Clinical Medicine and Clinical Research Center, UEF; Department of Psychiatry, Kuopio University Hospital, and Department of Eperimental Psychology, University of Oford. JoAnn E. Manson, MD, DrPH Pekka Mäntyselkä, MD, Professor of medicine and professor of women s health at Harvard Medical School, Chief of the Division of Preventive Medicine at Brigham and Women s Hospital in Boston. Co-PI of the Vitamin D and Omega-3 Trial in the USA. Professor of general practice. Institute of Public Health and Clinical Nutrition, UEF, Kuopio. Epertise in clinical and epidemiological studies about pain, Reporting 5

6 Sakari Suominen, MD, Tommi Tolmunen, MD, Matti Uusitupa, MD, cardiovascular risk factors, psychosocial distress and lifestyle. Adjunct professor of behavioral medicine, University of Turku. Professor of public health, University of Skövde Sweden. Epertise in epidemiological studies about the association and predictive power of various psychological and social factors on health outcomes, including perceived health and registry-based measures. Head of department, Kuopio University Hospital; Adjunct Professor of adolescent s psychiatry, University of Eastern Finland, Kuopio Emeritus professor of nutritional genomics and genetics, Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio. The core group (Table 3) possesses over 135 person-years of eperience in epidemiological research and supplementation studies. The group has received funding for the FIND study from the Academy of Finland in 2010 and from the University of Eastern Finland in 2011 with very high ratings (6/6 and 5.5/6, respectively). In addition to the FIND study, they have investigated associations between vitamin D and risk of major chronic diseases in the Kuopio Ischaemic Heart Disease Risk Factor Study (KIHD). In the KIHD we have shown that low serum 25(OH)D is associated with higher risk of mortality, type 2 diabetes and pneumonia, for eample. 16,24-26 In a recent RCT (VitDmet, Clinicaltrials.gov: NCT ) by the group, 71 pre-diabetic individuals were supplemented for 5 months with vitamin D 3 (same study protocol as in FIND to pilot) and demonstrated that the changes in the epression of VDR target genes both in peripheral blood mononuclear cells and adipose tissue samples correlated with alterations of the vitamin D status of the individuals However, only some 60% of the subjects responded on the level of their transcriptome profile and clinical and biochemical parameters to the vitamin D 3 supplementation. Collaboration The FIND is lead, organized and conducted by the Institute of Public Health and Clinical Nutrition, Faculty of Health Sciences, at the Kuopio Campus of the UEF, and various research groups at the campus are involved. The FIND study team includes investigators also from the University of Helsinki, University of Turku and from the National Institute for Health and Welfare in Finland. International collaboration is provided by Dr. JoAnn E. Manson from the Harvard Medical School, Dr. Elisabeth Bertone-Johnson from the University of Massachusetts, Dr. Adrian Martineau from the Queen Mary University of London and by Dr. Elina Hyppönen from the University of South Australia. Dr Manson is a co-pi of the Vitamin D and Omega-3 trial (VITAL) and a member of the FIND study team. Dr. Bertone-Johnson has long-time eperience in vitamin D research. Dr. Hyppönen is the leader of the D-CarDia collaboration ( National and international collaboration will be especially active in the future, when the data from the unique FIND trial becomes available. Infrastructure and environment The study is carried out at the Institute of Public Health and Clinical Nutrition ( within the School of Medicine, UEF Kuopio campus. Strengths of the Institute are the good facilities and the professional staff with long eperience both in large population-based cohort studies and in RCTs. The quality of research was evaluated in 2013 by international evaluators with a numeric evaluation of 5 (scale 1-6) (www2.uef.fi/fi/rae2013). Publication of study findings The highest quality open access journals or journals with an open access option are preferred for manuscript submission. Press releases and articles of the study findings will be actively submitted to the lay press and other media. The team members will also work closely with public health 6

7 authorities for application of the study findings in the general population. A website for the trial will also be created for active communication of the results of the study and applications for the public. VI TIMETABLE AND PROGRESS Study progress Recruitment has been closed and the total number of subjects in the study in September 2015 is Of the subgroup, 513 subjects remain in the study. The mailing of the year 3 study forms and the supplements for the year 4 have been started in September Note: The evaluation of the effect on the incidence of major chronic diseases (Objective 1) will become feasible only by a fairly long post-supplementation follow-up and pooling with other ongoing large supplementation studies. For instance, FIND is taking part in an individual patient data (IPD) meta-analysis of the large, ongoing vitamin D supplementation trials (VITAL, VIDA, TIPS-3, D-HEALTH and FIND). This task is currently lead and organised by Dr. Adrian Martineau from the Queen Mary University of London. Table 1. FIND study timeline BL mo mo mo mo mo Study forms - Food-frequency questionnaire Subgroup eaminations Blood samples - - Blood pressure Anthropometry DXA, muscle strength, balance Study visits (non-subgroup) DXA: dual-energy X-ray absorptiometry () 60 mo Table 2. Timetable for the research Task Study visits Measurements: Safety measurements (also s-glucose and lipids) Serum 25(OH)D Serum insulin Vitamin D binding protein Serum 1,25(OH) 2 D Statistical analyses Publication of key findings Reporting VII FINANCIAL PLAN The grant applied for from Finnish Cultural Foundation includes a part of the costs of the biochemical analyses (Table 4), including part-time salary for the chemist. Also included is a part of the salary for the study assistant and a grant for students. students will also apply for personal grants from other funding sources. Other goods and services include the printing and posting of the study forms and supplements, registry linkage to national health registries and publication costs of manuscripts. All PI s (Tuomainen, Voutilainen and Virtanen) are permanent employees of the UEF. They work on average 4-6 h/wk for the FIND project.

8 Table 3. Financial plan Research costs for the year 2016 (VAT included where applicable) Salaries (including overhead) 13,950 - Study assistant, 1 mo - Chemist 1.5 mo Grants for students, 2 mo 3,000 Consumables for the analyses 12,550 Other goods and services 13,000 Travel (study group meetings) 1,000 Overheads 15% 6,525 Total applied funding 50,025 Table 4. Upcoming measurements of the FIND subgroup participants Measurements Time of collection Year n Cost (incl. VAT) Serum 25(OH)D 2y, 5y ,200 Serum free and unbound BL, 6 mo, 1y 25(OH)D ,200 Serum insulin BL, 1y ,500 Serum insulin 2y, 5y ,300 Safety total calcium, ALAT, GGT, 5y creatinine and lipids ,000 Vitamin D binding protein BL, 1y ,900 Genes (vitamin D converting BL enzymes) ,000 Serum 1,25(OH)2D BL, 1y ,500 Serum 1,25(OH)2D 2y, 5y ,750 BL = baseline VIII EXPECTED SCIENTIFIC IMPACT OF THE RESEARCH There is increasing evidence from population studies about the potential health benefits of higher serum 25(OH)D concentrations, but so far there is no information from randomized placebocontrolled supplementation trials that does increasing 25(OH)D concentration through vitamin D supplementation actually have an effect on the risk of chronic diseases or other health outcomes. Because higher serum 25(OH)D concentration is also a good biomarker for a healthy lifestyle (i.e. low body mass inde, outdoor activities and fish consumption), only randomized supplementation trials with sufficiently high long-term doses can give an answer to this question. FIND is an ecellent study to investigate the health effects of vitamin D supplementation, because it is conducted in Finland, where UVB light is not sufficient to induce vitamin D formation in the skin most of the year. The use of two different doses of vitamin D in this supplementation study will give information about whether additional benefits are gained (without increased risk of side effects) with a higher dose or whether a lower dose is sufficient. Whether the results obtained in this study support the benefits of vitamin D supplementation or not, data from this trial will help to update public health guidelines and to make clinical recommendations and individual decisions. If vitamin D supplementation is found to have beneficial health effects (without marked side-effects), this supports larger fortification of foods with vitamin D and also recommendations for use of higher doses of vitamin D supplements, especially during periods of low eposure to summer sunlight. 8

9 IX References (own in bold) 1. Hilger J, et al. Br J Nutr 1-23 (2013). 2. Zittermann A, et al. Am J Clin Nutr 95, (2012). 3. Beveridge LA, Witham MD. Osteoporos Int 24, (2013). 4. Bouillon R, et al. J Clin Endocrinol Metab 98, E (2013). 5. Song Y, et al. Diabetes Care 36, (2013). 6. Bolland MJ, et al. The Lancet Diabetes & Endocrinology 24, (2014). 7. Chowdhury R, et al. BMJ 348, g1903 (2014). 8. Shipton EA, Shipton EE. Pain Res Treat 2015, (2015). 9. Carlberg C, Campbell MJ. Steroids 78, (2013). 10. Carlberg C. Front Physiol 5, 167 (2014). 11. IOM (Institute of Medicine). (2011). 12. Dastani Z, et al. Calcif Tissue Int 92, (2013). 13. Brannon PM. Scand J Clin Lab Invest Suppl 243, (2012). 14. Wagner D, et al. J Steroid Biochem Mol Biol 126, (2011). 15. Ghashut RA, et al. PLoS One 9, e92614 (2014). 16. Mursu J, et al. Eur J Epidemiol 30, (2015). 17. Chun RF, et al. J Steroid Biochem Mol Biol 144 Pt A, (2014). 18. Berry D, Hypponen E. Curr Opin Nephrol Hypertens 20, (2011). 19. Zhang Y, et al. Swiss Med Wkly 142, w13636 (2012). 20. Gozdzik A, et al. J Steroid Biochem Mol Biol 127, (2011). 21. Gandini S, et al. Adv Ep Med Biol 810, (2014). 22. Tuomainen TP, et al. J Diabetes Res 2015, (2015). 23. Hathcock JN, et al. Am J Clin Nutr 85, 6-18 (2007). 24. Virtanen JK, et al. Eur J Nutr 50, (2011). 25. Hurskainen AR, et al. Diabetes Metab Res Rev 28, (2012). 26. Aregbesola A, et al. J Epidemiol Community Health 67, (2013). 27. Carlberg C, et al. PLoS One 8, e71042 (2013). 28. Wilfinger J, et al. J Nutr Biochem (2014). 29. Ryynanen J, et al. Mol Nutr Food Res (2014). 9