Literature search and review related to specific preparatory work in the establishment of Dietary References Values for Copper (Lot 3) 1

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1 Supporting Publications 2012:EN-302 EXTERNAL SCIENTIFIC REPORT Literature search and related to specific preparatory work in the establishment of Dietary References Values for Copper (Lot 3) 1 Prepared by M. Bost (Edouard Herriot Hospital, Lyon), S. Houdart (ANSES), J.F. Huneau (AgroParisTech), E. Kalonji (ANSES), I. Margaritis (ANSES), M. Oberli (ANSES) ABSTRACT The objective of this literature search and was to identify the data from 1990 onwards upon which Dietary References Values (DRVs) for copper may be based. Articles were searched using three databases: Pubmed, Cochrane and Embase. Additional studies were identified by checking the reference lists of relevant s. Moreover, reports on DRVs from several countries were also considered as well as one study published in French relating to copper intakes in the French population. The search resulted in a total of 8004 references to screen on the basis of title and abstracts. 246 articles were then assessed from full text publications and 83 were finally included in this. Because of the lack of specific health outcomes related to copper intake, a large array of diseases was thus considered for the. Cardiac arrhythmia, cancer, arthritis, cognitive functions, respiratory disease and cardiovascular mortality were considered. No conclusion about the influence of copper intakes could be drawn from these studies. Studies identified also focused on copper status biomarkers as well as on copper metabolism and bioavailability, particularly its absorption and its excretion. Copper balance was sometimes calculated but only faecal and/or urinary copper losses were taken into account and these studies were therefore graded as at high risk of bias. Studies presenting only copper intakes in representative samples of national populations were included. Data concerning Cu intake of representative samples of healthy populations may indeed be useful to define an adequate intake for copper in case it would not be possible to estimate an average requirement due to lack of data, relevant criteria and/or good quality studies. Overall, the majority of studies were at high or moderate risk of bias and it seems that the evidence on which DRVs may be based for copper is poor. ANSES, 2012 KEY WORDS Copper, systematic, Dietary References Values (DRVs), biomarkers, status, health outcomes, endpoints. 1 Question No EFSA-Q Any enquiries related to this output should be addressed to nda@efsa.europa.eu Suggested citation: M. Bost (Edouard Herriot Hospital, Lyon), S. Houdart (ANSES), J.F. Huneau (AgroParisTech), E. Kalonji (ANSES), I. Margaritis (ANSES), M. Oberli (ANSES); Literature search and related to specific preparatory work in the establishment of Dietary References Values for Copper (Lot 3). Supporting Publications 2012:EN-302. [63 pp.]. Available online: European Food Safety Authority, 2012

2 SUMMARY This systematic was carried out preparatory to work by EFSA in order to establish Dietary References Values (DRVs) for copper (Lot 3 from the open call for tender CFT/EFSA/NUTRI/2011/01). This report summarizes the findings for copper. The literature was comprehensively searched from 1990 onwards for studies published in English, and only reports on copper intake levels below the tolerable upper intake (UL, 5 mg/d for adults) and on copper forms naturally present in foods or approved by the European Commission for use in foods or dietary supplements (Cupric carbonate, Cupric gluconate, Cupric citrate, Cupric sulfate, Copper lysine complexe) were considered. The search focused on studies in humans regarding maintenance of functional competence and the prevention of clinical deficiencies and chronic disease upon which DRVs may be based. Studies were included when copper intake and endpoints related to copper were measured (with the exception of studies measuring the usual copper intake in Caucasian population), as well as studies reporting copper levels in breast milk. Articles were searched using three databases: Pudmed, Cochrane and Embase. The search results were imported into Endnote and duplicate references were removed, which resulted in 7998 references to screen. One further article was identified by checking the reference lists of identified s and one study published in French about micronutrient intakes in the French population was also included. Reports on DRVs from several countries were also searched, resulting in 8004 references to screen on the basis of title and abstracts. 246 articles were assessed from full text publications, which led to the final inclusion of 83 articles. Relevant data (study details, methodology, results and validity and quality assessment) for each study were extracted into a Microsoft Excel document. Health endpoints identified were cardiac arrhythmia, cancer, arthritis, cognitive functions, respiratory disease and cardiovascular mortality. Studies focusing on copper metabolism and bioavailability were subdivided into studies calculating copper balance values and studies presenting copper absorption values. Cross-sectional studies and systematic reporting copper concentrations in breast milk were included as well as cross-sectional studies reporting copper intakes in representative samples of the European population. The majority of studies were at high or moderate risk of bias. Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 List of tables... 5 List of figures... 6 Background... 7 Terms of reference... 7 Acknowledgements... 8 Introduction and Objectives... 9 Introduction... 9 Objectives... 9 Materials and Methods... 9 Search strategy Inclusion for full text assessment Data extraction Validity assessment Results Validity assessment Health outcomes Cardiac Arrhythmia Other health outcomes Bioavailability and metabolism Biomarkers of copper status Plasma copper Ceruloplasmin Erythrocyte Superoxide Dismutase (SOD) Diamine oxidase (DAO) Skin lysyl oxidase Nutrient-nutrient interactions in various lifestage groups Biomarkers of immune response Biomarkers of bone metabolism Human milk concentrations Copper intakes in the European population Dietary references values in other countries (USA, UK, Australia/New Zealand, Nordic recommendations) Conclusion References list of articles included in the Appendix A: Study selection process flowchart Appendix B: Search strategies Appendix C: Table of references excluded at full-text articles screening Appendix D: Review protocol Enclosed document Appendix E: Extraction data Microsoft Excel spreadsheet for RCTs, CTs, Cohort, Balance and Cross sectional studies Enclosed document Appendix F: Assessment of internal validity in RCTs, observational studies and systematic s Appendix G: Extraction data Microsoft Excel spreadsheet for studies assessing copper intakes in the EU population Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

4 Appendix H: Extraction data Microsoft Excel spreadsheet for DRVs set in several countries Glossary / Abbreviations Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

5 LIST OF TABLES Table 1: Key reasons for classification at moderate or high risk of bias for each study design Table 2: Cardiac functions and copper intakes Table 3: Summary of main data to take into account in balance studies Table 4: Biomarkers of bone metabolism Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

6 LIST OF FIGURES Figure 1: Flow chart of the and selection process Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

7 BACKGROUND In 2005, The European Food Safety Authority (EFSA) received a mandate from the European Commission to the existing advice of the Scientific Committee on Food (SCF) published in 1993 on Dietary Reference Values (DRVs) for energy, macro- and micronutrients and other substances with a nutritional or physiological effect. While the setting of DRVs for macronutrients and energy has almost been finalized by the EFSA NDA Panel, the work on micronutrients started in 2010, and EFSA has launched call for tenders related to preparatory work in the establishment of DRVs for different nutrients. A tender related to preparatory work in the establishment of DRVs for some vitamins (A, C, E and K) and minerals (chromium, manganese, molybdenum, magnesium, potassium and fluoride) was awarded in 2010 (CFT/EFSA/NDA/2010/02). In 2011, a tender regarding preparatory work in the establishment of DRVs for further vitamins (thiamine, pantothenic acid, choline, vitamin B6, niacin and biotin) and for copper has been awarded (CFT/EFSA/NUTRI/2011/01). It is therefore required to carry out a comprehensive literature search and to identify health outcomes upon which DRVs may potentially be based for different life stages and gender groups of the general healthy population for micronutrients. Health outcomes comprise suitable indicators of nutrient adequacy used to define nutrient requirements, such as prevention of clinical deficiency symptoms, maintenance of nutrient-related functional competence, maintenance of cell (organ) integrity, achievement of sufficient nutrient body stores or status. In addition, if available, the scientific evidence on the relationship between dietary nutrient intake and risk of chronic disease, such as diabetes, cardiovascular disease or cancer should also be considered. The call for tender specified that the literature should be comprehensively searched for data published from 1990 onwards in order to update the scientific advice of the SCF dating from The literature search should focus on primary studies in humans reporting on the dose-response relationship between quantitative intakes of the nutrient within the physiological range and health outcomes on which DRVs may be based. TERMS OF REFERENCE Overall objective: The overall objective for Lot 3 of CFT/EFSA/NUTRI/2011/01 is to provide EFSA with a report containing an up-to-date of health outcomes upon which Dietary Reference Values could potentially be based for copper. Specific objectives: The specific objectives for Lot 3 of CFT/EFSA/NUTRI/2011/01 are as follows: - The contractor should carry out comprehensive literature searches to identify and retrieve all related information/data published in peer-ed journals in relation to the overall objectives of the contract. - The contractor should collate the data retrieved. - Data on the relevant micronutrient-related health outcomes should be further analyzed and a report should be prepared. The information should be transferred in a concise way to EFSA including the full list of references used for the micronutrient. References not considered pertinent should be listed and rationales should be provided for discarding these references. Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

8 ACKNOWLEDGEMENTS This contract was awarded by EFSA to: ANSES (Agence Nationale de Sécurité Sanitaire de l alimentation, de l environnement et du travail) Contract title: Preparation of an evidence report identifying health outcomes upon which Dietary References Values could potentially be based for copper Contract number: CT/EFSA/NUTRI/2011/04 Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

9 INTRODUCTION AND OBJECTIVES INTRODUCTION As the scientific advice on nutrient and energy intakes for the European Community by the Scientific Committee for Food (SCF, 1993) needs to be ed following a mandate from the European Commission to EFSA, this report on preparatory work focused on identifying information to derive DRVs for copper. This report only focused on copper forms naturally present in foods or those approved by the EC (EC directive 2002/46/EC; EC Regulation No 1925/2006) for use in foods or foods supplements. This report will focus on levels below the tolerable upper intake (UL) set at 5 mg/d for adults (SCF, 2003). Because of the lack of specific health outcomes related to copper intake, a large array of diseases was thus considered for the. Cardiac arrhythmia, cancer, arthritis, cognitive functions, respiratory disease and cardiovascular mortality were considered. No conclusion about the influence of copper intakes could be drawn from these studies. Different biomarkers (plasma Cu, ceruloplasmin, erythrocyte SOD, Cu chaperone for SOD, platelet cytochrome-c oxidase, plasma diamine oxidase, skin lysyl oxidase, peptidyl glycine α-amidating monooxygenase, proteomic/transcriptomic biomarkers) have been proposed to assess Cu status (Eurreca, 2011). Most of the time, these markers have a poor sensitivity and/or specificity and there is not enough data to draw firm conclusions about their usefulness as biomarkers of copper status (Harvey et al., 2009). However, despite the limitations reported in the above mentioned quantitative s, these biomarkers were included in the present evaluation. Identified studies also focused on copper metabolism and bioavailability, particularly its absorption and its excretion. Copper balance was sometimes calculated in these studies. Studies presenting only copper intakes in representative samples of national populations were included as well. Data concerning Cu intake of representative samples of healthy populations may be useful to define an adequate intake for copper in case it would not be possible to estimate an average requirement due to lack of data, relevant criteria and/or good quality studies. OBJECTIVES The aim of this literature search and was to collect existing and relevant scientific evidence and identify health outcomes upon which DRVs may potentially be based for copper in the context of a balanced diet for the healthy European population. The search was based on a systematic approach and relevant articles identified were tabulated in an Excel Microsoft datasheet. In order to minimize potential reporting bias, this was performed through a comprehensive and reproducible search using a clearly defined selection and reporting protocol (Appendix D). MATERIALS AND METHODS The materials and methods used for this systematic are detailed in the protocol presented in Appendix D. Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

10 SEARCH STRATEGY In January and February 2012, electronic searches were carried out following definition of the search strategy (Appendix B). The databases searched were Pubmed, Cochrane and Embase. Additional references were identified by checking the reference lists of relevant s and articles identified by the literature search. Several key DRVs reports were also used. Search results were imported into Endnote, duplicates were removed which resulted in 8004 articles to screen for relevance to the key element of the research question on the basis of titles and abstracts. This screening enabled to exclude articles that were obviously irrelevant according to some pre-defined criteria: non-english language studies, articles not concerning copper, articles studying copper administration other than orally etc. Only original articles or systematic s were taken into account (non-systematic s, conference abstracts, letters etc were excluded. The reference lists of non-systematic s were checked). A study was included at the title and abstract screening if its relevance was unclear at this stage. Articles included following the title and abstract screening were retrieved in full-text. INCLUSION FOR FULL TEXT ASSESSMENT Full-text articles retrieved were evaluated for inclusion according to pre-defined criteria in the protocol (Appendix D). The exclusion criteria used were: - articles published before 1990; - articles published in another language than English; - non-systematic s, conference abstracts letters, case-studies; - animal, in-vitro trials; - irrelevant population (considered not to be healthy, e.g. obese people; athletes) - absence of copper intake assessment; - supplements not orally ingested; - multi supplementations (which does not enable to isolate effects of copper); - supplements not authorized in foods or dietary supplements (Regulation EC n 1925/2006 on the addition of vitamins and minerals and of certain other substances to foods); - studies in which all copper intakes were higher than the UL of 5 mg/d set for adults; - toxicity research (high copper concentrations in water); - no relationship between intake and status, intake and health (except studies reporting copper levels in milk and studies reporting intakes in samples representative of a population). The full text assessment of included articles was carried out with a minimum of 10% duplication. When the two ers disagreed, the paper was discussed within the project group to reach a consensus on inclusion. Prior to data extraction, the following additional criteria were used: - studies without a suitable control or baseline measure; Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

11 - relevance to the European context in terms of diet, lifestyle and ethnicity of the population group studied; - studies not accurately quantifying copper dose or measuring a change in biomarker status to reflect change in copper intake. Studies excluded were tabulated and the reason for exclusion was reported (Appendix C). DATA EXTRACTION Data from included studies were extracted into a Microsoft Excel (Appendix E) spreadsheet and 10 % was checked by a second er for completeness and accuracy. Data extraction included: - study details: study design, study references, study population characteristics (age, type and number of participants, country of origin), measures of intake, status and outcomes, biomarkers measured, aim of the study; - study methodology: confounding factors identified, dietary intake estimation method and validity, compliance and dose check, analytical methods of measures of biomarkers etc ; - study results: numerical relationship(s) for the relevant intake-status-health associations, or other relevant data; - validity assessment: details of study quality and conclusion on the risk of bias (see below). VALIDITY ASSESSMENT Study validity criteria were collated into a Microsoft Excel spreadsheet (Appendix E), during the data extraction phase. The criteria were then assessed according to study methodology using a scheme devised by the EURRECA Network of Excellence (Appendix F). Studies were classified as high, moderate, low or unclear risk of bias according to each study methodology. Key criteria were: - SR: databases searched, inclusion criteria, validity assessment; - RCT: randomisation, allocation method, blinding, loss to follow up; - Cohort, case-control: similarity of groups at baseline, potential confounders (and their adjustment), intake assessment methods; - Cross-sectional: exposure and status assessment, potential confounders; - Case-study: number of cases explored and strength of measured change; - Balance: background exposure, measurement of dose provided. Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

12 RESULTS After removal of duplicates, 7998 articles to screen on the basis of titles and abstracts were identified through databases searching. One other article was identified by checking the reference lists of relevant s. Key reports on DRVs were also considered, which resulted in 8004 articles to screen against pre-defined inclusion criteria. Following this step, 246 articles were retrieved for fulltext assessment and 83 articles were finally included for the. This is summarized in the following chart (Figure 1): Table 1: Figure 1: Flow chart of the and selection process PUBMED, COCHRANE, EMBASE : N=7998 Articles identified from other sources: N=6 Endnote Library : N= 8004 Articles screened on the basis of title and abstract after duplicates elimination 246 full-text articles assessed for eligibility. 83 articles included in the The study details, methodology, results and quality are described by endpoint in the subsections below. Main data for some endpoints are presented in tables 2, 3 and 4 while data extraction is tabulated in full in Appendix E. VALIDITY ASSESSMENT Validity assessment was completed for each included article. Articles were assessed against defined criteria and classified as at low, moderate, high or unclear risk of bias (details of the validity assessment procedure are provided in Appendix F). Key reasons for assessment of articles at unclear, moderate or high risk of bias are summarised by study type below (Table 1). Out of the 2 systematic s (SR) included, one was judged as at high risk of bias and one as at low risk of bias. Nineteen controlled trials (CT) were graded as being at high risk of bias, 3 at moderate and 5 at unclear risk of bias. Seven balance studies were judged as being at high risk of bias, 2 as at moderate risk of bias, 2 as at low risk of bias and the risk of bias was unclear for one study. The five cohort studies were graded as being at low risk of bias. The 2 case-controls studies included were graded as being at high risk of bias. Ten cross-sectional studies were at high risk of bias, one at Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

13 moderate risk of bias, two at low risk of bias, while the risk of bias was identified as unclear for 4 of them. The assessment is tabulated in full by study type in Appendix E. Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender

14 Table 1: Key reasons for classification at moderate or high risk of bias for each study design Moderate risk High risk Unclear risk Controlled trials No adequate report of background dietary exposure. Not randomized and/or no adequate blinding. Randomized without adequately reporting the method of randomization, allocation concealment or blinding. No measure of background intakes. Randomized but unclear information about sequence generation and allocation concealment and/or unclear blinding method. Dropout not adequate or/and outcome data incomplete. Balance studies Some factors were not adjusted for. No assessment of faecal and urinary and sweat and integumentary losses (or sweat and integumentary losses not taken into account in balance calculation). Cu absorption not measured in all subjects. No clear information enabling to decide if study dealt with confounding factors adequately and it was unclear if the study included a representative group of those approached to participate. No adequate assessment of copper intake. Cross-sectional studies / No adequate assessment of copper intake. No adequate report of adjustment for confounders. No clear information enabling to decide if study dealt with confounding factors adequately and unclear assessment of exposure. Cohort and case control studies % of those approached participating and similarity of participants and non-participants unclear. No adequate report of adjustment for confounders. Assessment of exposure not adequate. / Systematic s / No adequate report of study inclusion criteria, study assessment, characteristics and pooling. / Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the

15 HEALTH OUTCOMES Symptoms accompanying severe copper deficiency have been reported during total parenteral nutrition or after gastric bypass surgery and include normocytic and hypochromic anemia, hypercholesterolaemia, skin and hair hypopigmentation, leukopenia, neutropenia, myelodisplasia and in the majority of patients neurological findings, most commonly due to neuromyelopathy (human swayback). Osteoroposis and scoliosis have also been reported in copper-deficient infants and children (FNB, IOM, 2001; Klevay et al., 2011). Marginal copper deficiency may lead to oxidative stress and cognitive decline while mild copper excess may trigger gastrointestinal disorders and liver damages. Because of the lack of specific health outcomes related to copper intake, since copper is involved in numerous cuproenzymes with wide range of functions (Appendix D), a large array of diseases was thus considered for this. Cardiac Arrhythmia An increased occurrence of ventricular premature discharges (VPDs) during copper depletion has been reported in two controlled trials carried out on postmenopausal women (Milne et al., 1996 and 2001) (Table 2 and Appendix E). In the first trial, a significant increase in the number of ventricular premature discharge (VPDs) was observed in three women out of 13 after 21, 63 and 91-d on a low copper diet (0.57 mg/d for 105 days). All women involved in the trial consumed the same diet (consisting in an equilibration period with 1.37 mg Cu/d for 35d, a deprivation period with 0.57 mg Cu/d for 105d and a repletion period with 2 mg Cu/d for 35d). However, the author does not provide any information regarding the results of the electrocardiogram or the extent of the increase in VPDs. No significant increase was observed for the other women consuming this low copper diet (Milne et al., 1996). In the 2001 study, 3 women out of 12 on a low copper diet (1 mg/d during a 90-d controlled period) exhibited abnormal electrocardiographic recording (premature ventricular discharge) requiring copper supplementation before the end of the study. However, two of these women still exhibited an increased number of abnormal ventricular discharge after Cu supplementation (the duration of Cu supplementation is not specified). Moreover, none of the women receiving 3 mg Cu/d showed significant changes in their electrocardiograms (Milne et al., 2001). The results of heart rate (HR) monitoring are also reported in a third study examining the effect of Cu depletion in young men (Turnlund et al., 1997). In this study, no difference was observed in the occurrence of VPDs and supraventricular ectopic beats (SVEs) between the adaptation, depletion and repletion periods. Due to lack of information regarding randomization and blinding, these three studies are at high risk of bias and no clear conclusion can be drawn regarding the relationship between dietary Cu and cardiac arrhythmia. Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender considered as an output adopted by the Authority. The European food Safety Authority reserves its rights, view and position as regards the

16 Table 2: Cardiac functions and copper intakes (Cf Appendix E) Study references n Age Sex Intervention and control or exposure Dietary intake estimation Follow up time Results related to cardiac functions Risk of bias Low dietary zinc alters indices of copper function and status in postmenopausal women. Milne, y (±6.7) F Low Cu (n=14) Equilibration periods : Cu=2mg/d) Controlled period : Cu=1 mg/d with low Zn (first 90-d) and high Zn (last 90-d) High Cu (n=14) Equilibration periods : Cu=2mg/d) Controlled period : Cu=3 mg/d with low Zn (first 90-d) and high Zn (last 90-d) Fixed diets low in Cu (0.60mg/d) + CuSO4 solution to achieve the desired level of Cu 10-d equilibration period, 2*90-d controlled period separated by a 2 nd 10-d equilibration period 3 women out of 12 on low Cu exhibited abnormal electrocardiographic recording. Cu-balance was more sensitive to changes in dietary Zn than changes in dietary Cu. High Effects of a diet low in copper on copper-status indicators in postmenopausal women. Milne, y (±8.8) F 3 successive levels of Cu for all subjects: Equilibration : 1.37 mg/d Depletion : 0.57 mg/d Repletion : 2.57 mg/d Fixed diet, low in Cu (0.57 mg/d) + CuSO4 solution to achieve the desired level of Cu Equilibration = 5 w Depletion = 15 w Repletion = 4 w Significant increase over control values in the number of ventricular premature discharge in three women after 21, 63 and 91 days of consuming low Cu diets. High Copper status of young men consuming a lowcopper diet. Turnlund, ±4 y M Equilibration: Cu = 0.66 mg/d Depletion: Cu = 0.38 mg/d Repletion: Cu = 2.49 mg/d Fixed diet, low in Cu (0.38mg/d). Cu content of the diet adjusted to reach 0.66 mg and 2.49 mg/d by adding a CuSO4 solution to the formula drink of each meal Equilibration= 24 d Depletion= 42 d Repletion= 24 d HR monitoring revealed that several subjects had a number of PVCs and SVEs before, during and after the Cu-depletion period. No significant difference in the number of events of either PVCs or SVEs at the different time points and no change during the depletion period. High Supporting publications 2012:EN by the author(s) in the context of a contract between The European Food Safety Authority and the author(s), awarded following a tender considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the

17 Other health outcomes Cancer, respiratory and cardiovascular disease mortality, arthritis and cognitive functions were mostly studied throughout cohort or cross-sectional studies. I. Cognitive decline A cohort study of 3718 males and females by Morris et al (2006) identified a potentially adverse effect of Cu-containing supplements on cognitive functions in the context of high-saturated trans fatty acid intakes (but not when the diet was low in saturated and trans fatty acid) (cf Appendix E). This cohort study, graded as being at unclear risk of bias, does not enable to draw conclusion on the influence of copper intakes on cognitive decline since other factors than copper intakes are involved. II. Cancer In a randiomized controlled trial (RCT) involving 17 men, a low-cu diet (0.59 mg Cu/d) given for 6 weeks was associated with lower Cu concentrations in faecal water, higher faecal water alkaline phosphatase activity and higher in vitro production of free radical in faeces compared to an adequate Cu-diet (2.59 mg Cu/d for 6 weeks), which suggests that a low Cu diet is associated with a less favourable faecal environment (higher free radical generation and alkaline phosphatase activity, higher cytotoxic activity) which might explain higher colon cancer susceptibility in Cu-deficient subjects (Davis et al., 2003) (cf Appendix E). Two cohort studies, one cross-sectional study and one case-control study assessed the association between copper intake/status and risk for several types of cancer (cf Appendix E). Mahabir et al (2010) showed no association between total Cu intake and lung cancer risk in a cohort study of subjects in the USA: the subjects were mostly white (91 %), 59.7 % of the subjects were men, 49 % of the participants were former smokers, 12 % were current smokers and 36 % never smoked (smoking status was unknown for 3 % of the subjects). Thompson et al (2010) did not identify an association between total or dietary Cu and the risk for Non-Hodgkin s lymphoma, diffuse large B-cell lymphoma or follicular lymphoma in a cohort study of women. A case-control study (261 cases and 261 controls in Milano and Montpellier) by Cavallo et al. (1990) evaluated the association between copper intake and status and breast cancer risk in women aged between 25 and 65 years old. In Milano the mean age of the subjects was 49.6±8.5 years for cases and 47.1±9.4 years for controls. In Montpellier, the mean age was 53.1±9.8 years for cases and 53.1±8.6 years for controls. The Odds Ratio for breast cancer did not show a significant trend among quartiles of copper intake (Milano) or serum Cu (Montpellier and Milano) (cf Appendix E). This study graded at high risk of bias did not suggest that Cu might be associated with breast cancer. Dabek et al. (1994) carried out a cross-sectional study with 23 females with breast cancer, 23 vegetarian and 25 omnivorous controls. They observed that the pre-menopausal breast cancer group had significantly higher plasma Cu levels than the pre-menopausal controls (cf Appendix E). However, because of its observational nature, this study graded as being at high risk of bias does not enable to establish a cause and effect relationship. Thus, according to these studies, no conclusion can be drawn regarding the health outcome cancer. III. Respiratory and cardiovascular disease mortality One cohort study of 1054 subjects aged 65 and over (mean age 76.6±7.4 years, 49% females) judged as being at low risk of bias evaluated the predictive significance, in terms of subsequent mortality, of biochemical indices for nutrients mediating redox-modulatory functions in living tissue (Cu, Zn, Se, Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender

18 vitamins A, C, E, carotenoids) (Bates et al., 2011). Plasma copper concentration was a significant predictor of all-cause mortality, cardiovascular mortality but not cancer or respiratory disease mortality when adjusted for age and sex. When subdivided by sex, the predictive power of plasma Cu was only significant for men. However, dietary Cu intake was not a predictor of mortality (cf Appendix E). IV. Arthritis One cohort study at low risk of bias carried out on female subjects (mean age 61.4 years) (Cehran et al., 2003) found no association between total (diet and supplements) or dietary Cu intake and risk of rheumatoid arthritis. There was a weak but significant inverse association between the use of Cu supplements and the risk of rheumatoid arthritis, which did not persist after further adjustment for confounders (age and energy intakes, other risk factors) (cf Appendix E). V. Blood pressure Hajjar et al (2000) described the association between blood pressure (systolic (SBP) and diastolic blood pressures (DBP)) and copper intake (as well as intake of several other nutrients) in a crosssectional study involving subjects (mean age 47.6±0.2 years) in the USA. For this, the USA were divided into four regions (14% of the subjects were from northeast, 19% from midwest, 43% from south and 24% from west) and comparisons between the four regions were performed. In this study judged as being at low risk of bias, the south region had the highest SBP and DBP (P<0.005) and reported the lowest copper intake, but also the least fibre, potassium, calcium, phosphorous, magnesium, riboflavin, niacin, iron, vitamins A, C and B-6 intakes and the highest consumption of sodium, monounsaturated fatty acids, polyunsaturated fatty acids and cholesterol (cf Appendix E). VI. Lipoproteins A cross-sectional study at high risk of bias of Bo et al. (2008), carried out on 1197 apparently healthy subjects (no use of prescription medicine, without diabetes, cardiovascular disease, dyslipidemia) from both sexes, observed a negative association between dietary or serum Cu and total and LDLcholesterol, suggesting that high copper intake and status is associated with a better metabolic profile. A second cross-sectional study with 189 participants (without diabetes, cardiovascular disease, hypotension) judged as at high risk of bias showed that serum copper was positively associated with HDL (Ghayour-Mobarhan et al., 2005) (cf Appendix E). In a RCT (for which risk of bias was unclear) of Davis et al. (2003), total and LDL cholesterol did not differ between the low (0.59 mg Cu/d) and adequate Cu (2.59 mg Cu/d) periods (6 weeks each). In the RCT of Medeiros et al (1991) graded as being at moderate risk of bias, supplementation with 2 mg Cu/d for 6 weeks triggered an increase in total serum LDL cholesterol at 4 weeks while VLDL cholesterol declined (in comparison with the placebo group). The percentage of cholesterol as LDL increased at 6 weeks of supplementation compared to the initial baseline value, while the percentage of cholesterol as VLDL decreased compared to the baseline value in the supplemented group (cf Appendix E). BIOAVAILABILITY AND METABOLISM A total of 14 studies addressing Cu absorption, excretion and retention were selected in the process. Five out of these 14 studies report estimation of Cu balance, with 4 assessing balance at different levels of Cu intake and one addressing the effect of polyphenol-rich beverages on Cu balance. One additional study provides information regarding faecal and urinary Cu excretion at Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender

19 different levels of intake but does not report estimation of Cu balance. The remaining 6 studies report data related to Cu absorption at different ages and in different dietary contexts. Balance calculation requires a thorough assessment of nutrient losses and none of the 5 balance studies met this criterion. Therefore, they must be considered as being at high risk of bias, although dietary intake was usually carefully controlled. In only one study were faecal, urinary and sweat and integumentary losses measured in some subjects (Milne et al., 1990). In this study, however, sweat and integumentary losses were not taken into account in the final balance calculation. Balance calculation took urinary losses into account in 3 studies, 2 of them comparing balances at different levels of dietary Cu (Milne et al., 1990; Turnlund et al., 2005), and only faecal losses were considered in the remaining studies (cf Table 3 and Appendix E). The digestive tract is the main route of Cu excretion. Faecal Cu losses increase almost linearly with dietary intake and range from 0.33 mg/d for a dietary Cu intake of 0.38 mg/d (Turnlund et al., 1998) to 2.17 mg/d for a dietary Cu intake of 2.49 mg/d (same study). In contrast, urinary Cu shows little or no variation with dietary Cu intake and ranges from 11 µg/d (Turnlund et al., 1998) to 60 µg/d (Milne et al., 2001). The only data available regarding sweat and integumentary losses suggest that these are close to µg/d in adults (Milne et al., 1990). At copper intakes above the UL of 5 mg/d (6 and 7.8 mg/d) and at 2.49 mg/d, Cu balance was reported to be positive. Balance values were negative for copper intakes from 0.38 to 0.69 mg/d, while equilibrium was reached at a copper intake of 1.6 mg/d (cf Table 3 and Appendix E). However, because urinary losses were seldomly considered and integumentary losses were never taken into account, these results must be examined with caution. Moreover, a further limitation of some of the balance studies selected during the process (Prystai et al., 1999; Turnlund et al., 1998; Turnlund et al., 2005) is the short duration of the periods during which dietary Cu is maintained at a fixed level before balance measurement (15 to 18 d), which may be insufficient for homeostatic adaptation to occur. Seven studies only provide estimations of copper absorption (%), at different ages or with different diets. In breast-fed infants (1-3 months), apparent Cu absorption ranged from 75 to 83% and was unaffected by Cu supplementation in a study judged as being at high risk of bias (Olivares et al., 2002). Apparent Cu absorption was still very high (75%) in pre-school children (1-4 y) consuming a diet typical of US children in a study graded as being at moderate risk of bias (Griffin et al., 2007). In adults, apparent Cu absorption is much lower, typically in the 20-40% range (Harvey et al., 2005; Hunt et al., 1998; Hunt et al., 2001; Ducros et al., 2005). In two studies, the faecal excretion of absorbed Cu was taken into account to estimate true Cu absorption, which ranged from 45 to 49% (Harvey et al., 2003 and 2005). The included studies presented above thus show that true Cu absorption seems to be constant for a dietary Cu level ranging from 0.7 to 6 mg/d. The effect of diet quality on Cu absorption has been examined in three studies. In adults, apparent Cu absorption (in %) tended to be higher with omnivorous diets than with lactoovovegetarian diets (Hunt et al., 1998 and 2001). However, this difference was compensated for by the higher Cu-content of the lactoovegatarian diets compared to the omnivorous diets, resulting in similar amounts of Cu absorbed daily (cf Table 3 and Appendix E). These two studies are at low or moderate risk of bias. In another study, a small but significant increase in apparent Cu absorption was observed when short-chain fructo-oligosaccharides (sc-fos) were added to the diet compared to placebo (Ducros et al., 2005). However, this last study is at high risk of bias and the nutritional significance of this increase is questionable. Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender

20 Table 3: Summary of main data to take into account in balance studies (cf Appendix E) Study reference Cu losses measured Study duration Dietary copper Copper balance (mg/d) Risk of bias Long-term high copper intake: effects on copper absorption, retention, and homeostasis in men. Turnlund JR, Faecal and urinary Cu Cu = 1.6 mg/d : 18 d Cu = 7.8 mg/d : 18 d (after 129-d of a high-cu diet (9.6 mg/d)) 1.6 mg/d mg/d 0.67 High Copper absorption, excretion, and retention by young men consuming low dietary copper determined by using the stable isotope 65Cu. Turnlund JR, Faecal Cu Cu = 0.66 mg/d : 24 d Cu= 0.38 mg/d : 42 d Cu= 2.49 mg/d : 24 d 0.66 mg/d mg /d mg/d High Adaptive responses in men fed lowand high-copper diets. Harvey LJ, 2003 Faecal Cu Cu = 1.6 mg/d : 56 d Cu = 0.69 mg/d : 56 d Cu = 6 mg/d : 56 d 1.6 mg/d 0.00 ± 0, mg/d ± mg/d 0.75 ± 1.05 High Effect of copper intake on balance, absorption, and status indices of copper in men. Milne DB, 1990 Faecal, urinary, sweat, integumentary Cu Cu = 1.29 mg/d : d Cu = 0.89 mg/d : d Cu= 5.05 mg/d : d 1.29 ± 0.44 mg/d ± ± 0.09 mg/d ± ± 0.72 mg/d 0.51 ± 0.30 High Calcium, copper, iron, magnesium, and zinc utilization of humans as affected by consumption of black, decaffeinated and green teas. Prystai EA, 1999 Faecal and urinary Cu 56-d study: 4 treatments of 14-d each Control Dietary Cu= 1.2 mg/d -0.1 ± 0.1 Black tea Dietary Cu= 1.3 mg Cu/d -0.4 ± 0.1 Decaffeinated black tea Dietary Cu= 1.3 mg/d -0.4 ± 0.1 Green tea Dietary Cu= 1.4 mg/d -0.1 ± 0.1 High Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the

21 High Cu 47.8 µmol/d (Low Zn) -0.8 µmol/d Low dietary zinc alters indices of copper function and status in postmenopausal women. Milne DB, Faecal and urinary Cu 200 d High Cu 48.0 µmol/d (High Zn) Low Cu 16.2 µmol/d (Low Zn) 2.8 µmol/d µmol/d High Low Cu 16.4 µmol/d (High Zn) -1.6 µmol/d Intestinal absorption and losses of copper measured using 65Cu in zincdeprived men. Taylor, 1991 Faecal Cu 58 d mg Cu/d mg Zn/d mg Zn/d Positive Cu balance High Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the

22 Study reference Cu losses measured Study duration Copper doses Apparent Cu absorption (%) Risk of bias Influence of Short-Chain Fructo- Oligosaccharides (sc-fos) on Absorption of Cu, Zn, and Se in Healthy Postmenopausal Women. Ducros, Faecal Cu Two 35-d periods: usual diet from d-1 to d-22 and controlled diet from d-23 to d ±0.08 mg/d with sc-fos 26.8±5.3 to 29.5±4.7 with placebo 21.7±3.7 to 28.9±5.6 p=0.042 Unclear Use of mathematical modelling to study copper metabolism in humans. Harvey LJ, Faecal and urinary Cu Urinary Cu measured over 7 d and faecal Cu measured over 14d 1.4 ± 0.4 mg/d (Usual Cu intake) 33± 3 Low Apparent copper absorption from a vegetarian diet. Hunt JR, 2001 Faecal Cu Apparent Cu absorption measured after 4 wk of diet Lactoovovegetarian diet 1,45 mg Cu/9,2 MJ Nonvegetarian diet 0,94 mg Cu/9,2 MJ ,1 Low Zinc absorption, mineral balance, and blood lipids in women consuming controlled lactoovovegetarian and omnivorous diets for 8 wk. Hunt JR, Faecal Cu 8-w nonvegetarian and 8-w lactoovogetarian diets. Total duration = 16 weeks. Lactoovovegetarian diet 2.5 ± 1.8 mg/d Nonvegetarian diet 1.5 ± 1.2 mg/d 18 ± ± 15 Moderate Zinc homeostasis in 1-4 year olds consuming diets typical of US children. Griffin IJ, Faecal Cu Adaptation period: 7 d. supplementation Absorption measurement: 5 d No 408 ± 116 µg/d 75.1 (10.8) Moderate Age and copper intake do not affect copper absorption, measured with the use of 65Cu as a tracer, in Faecal Cu Supplementation = 15 d Balance : 72 h Supplemented (µg Cu/kg/d) 1 mo 86.3 ± (9.1) 3 mo 55.9 ± (11.3) High Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the

23 young infants. Olivares M, Unsupplemented (µg Cu/kg/d) 1 mo 60.3 ± (5.8) 3 mo 35.6 ± (15.2) 23.3 µmol/d Faecal Cu 17.2±1.3 Urinary Cu 0.63±0.31 µmol/d Effects of a diet low in copper on copper-status indicators in postmenopausal women. Milne DB, Faecal and urinary Cu 5w: 1.37 mg/d 15w : 0.57 mg/d 4w : 2.57 mg/d 9.0 µmol/d Faecal Cu 6.0±1.1 Urinary Cu 0.47±0.31 µmol/d High 42.0 µmol/d Faecal Cu 31.7±5.2 Urinary Cu 0.47±0.31 µmol/d Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the

24 BIOMARKERS OF COPPER STATUS The EURRECA work (2011) carried out on biomarkers of status and exposure for several vitamins and minerals established that no conclusions could be drawn on the usefulness of serum Cu, ceruloplasmin, erythrocyte Cu, Cu-Zn-superoxide dismutase (SOD), plasma diamine oxidase (DAO), CCS (Cu chaperone for SOD), skin lysyl oxidase and peptidyl glycine α-amidating monooxygenase (PAM) activity, as biomarkers of copper status (lack or insufficient data or no relevant data identified). Only total ceruloplasmin protein was shown, in depleted individuals, to be related to copper status and plasma copper was identified as the currently most useful biomarker available for assessing copper status at the population level. In adults Plasma copper Ten controlled trials reporting the effect of dietary Cu intake on plasma Cu were identified in our search. In one of these trials judged as at high risk of bias, a small but significant decrease in plasma Cu was observed in young healthy men at the end of a Cu depletion period (dietary Cu=0.38 mg/d, plasma Cu=12.6 µmol/l) compared to the preceding equilibration period (dietary Cu=0.66 mg/d, plasma Cu=13.8 µmol/l) or the subsequent repletion period (dietary Cu=2.49 mg/d, plasma Cu=13.6 µmol/l) (Turnlund et al, 1997; Werman et al., 1997) (cf Appendix E). In the other trials, the range of dietary Cu was mg/d (Milne et al., 1996), mg/d (Davis et al, 2003), mg/d (Harvey et al., 2003), mg/d (Baker et al., 1999), (Turnlund et al., 1990), mg/d (Milne et al., 1990), mg/d (Araya et al., 2003), 1-3 mg/d (Milne et al., 2001). In none of these studies did the authors report any difference in plasma Cu between the different dietary Cu periods (cf Appendix E). Therefore, it seems that plasma Cu does not vary for daily intakes between 0.57 mg and the UL. A very low copper intake (0.38 mg/d) may result in a decreased plasma Cu but this remains questionable as such a low intake was imposed in only one trial. Moreover, this trial is at high risk of bias because of the absence of randomization. Ceruloplasmin As for plasma Cu, plasma ceruloplasmin and ceruloplasmin activity significantly declined when dietary copper intakes shifted from marginal (0.66 mg/d for 24 days) to low (0.38 mg/d for 42 days) in one controlled trial involving male subjects (Turnlund et al., 1997; Werman et al., 1997). However, plasma ceruloplasmin and ceruloplasmin activity did not increase after a repletion period providing 2.49 mg Cu/d for 24 days. As previously mentioned, this trial is at high risk of bias because of the absence of randomization (cf Appendix E). No effect of dietary Cu on plasma ceruloplasmin was reported in the other trials identified during our search (Milne et al., 1990, 1996, 2001; Baker et al., 1999; Turley et al., 2000; Kehoe et al, 2000; Harvey et al., 2003; Davis et al., 2003; Araya et al., 2003; Turnlund et al., 2004) (cf Appendix E). Erythrocyte Superoxide Dismutase (SOD) The effect of dietary Cu on the activity of the Cu/Zn erythrocyte SOD has been assessed in 5 controlled trials and one balance study. The risk of bias of the study from Davis et al. (2003) was unclear while the studies of Harvey et al (2003), Kehoe et al (2000), Milne et al (2001) and Turnlund et al (1997) were judged as at high risk of bias (cf Appendix E). Supporting publications 2012:EN by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender