An international workshop of invited experts and partners in the EC-funded Network of Excellence, EURRECA (www.eurreca.org) was held in Norwich on 18–20 February 2008 to discuss biomarkers of micronutrient status.

Four methods are recommended for assessment of iodine nutrition: urinary iodine concentration, the goitre rate, and blood concentrations of thyroid stimulating hormone and thyroglobulin. These indicators are complementary, in that urinary iodine is a sensitive indicator of recent iodine intake (days) and thyroglobulin shows an intermediate response (weeks to months), whereas changes in the goitre rate reflect long-term iodine nutrition (months to years). Spot urinary iodine concentrations are highly variable from day-to-day and should not be used to classify iodine status of individuals. International reference criteria for thyroid volume in children have recently been published and can be used for identifying even small goitres using thyroid ultrasound. Recent development of a dried blood spot thyroglobulin assay makes sample collection practical even in remote areas. Thyroid stimulating hormone is a useful indicator of iodine nutrition in the newborn, but not in other age groups. For assessing iron status, haemoglobin measurement alone has low specificity and sensitivity. Serum ferritin remains the best indicator of iron stores in the absence of inflammation. Measures of iron-deficient erythropoiesis include transferrin iron saturation and erythrocyte zinc protoporphyrin, but these often do not distinguish anaemia due to iron deficiency from the anaemia of chronic disease. The serum transferrin receptor is useful in this setting, but the assay requires standardization. In the absence of inflammation, a sensitive method to assess iron status is to combine the use of serum ferritin as a measure of iron stores and the serum transferrin receptor as a measure of tissue iron deficiency.

The essentiality of copper (Cu) in humans is demonstrated by various clinical features associated with deficiency, such as anaemia, hypercholesterolaemia and bone malformations. Despite significant effort over several decades a sensitive and specific Cu status biomarker has yet to be identified. The present article updates a comprehensive review recently published by the authors which assesses the reliability and robustness of current biomarkers and outlines the on-going search for novel indicators of status(1). The essential features of this earlier review are reiterated whilst considering whether there are other approaches, not yet tested, which may provide valuable information in the quest for an appropriate measure of copper status. Current biomarkers include a range of cuproenzymes such as the acute phase protein caeruloplasmin and Cu-Zn-superoxide dismutase all of which are influenced by a range of other dietary and environmental factors. A recent development is the identification of the Cu chaperone, CCS as a potential biomarker; although its reliability has yet to be established. This appears to be the most promising potential biomarker, responding to both Cu deficiency and excess. The potential for identifying a ‘suite’ of biomarkers using high-throughput technologies such as transcriptomics and proteomics is only now being examined. A combination of these technologies in conjunction with a range of innovative metal detection techniques is essential if the search for robust copper biomarkers is to be successful.

The role of zinc deficiency as an important cause of morbidity and impaired linear growth has prompted the need to identify indicators of population zinc status. Three indicators have been recommended – prevalence of zinc intakes below the estimated average requirement (EAR), percentage with low serum zinc concentrations, and percentage of children aged < 5 years who are stunted. This review outlines steps to estimate the prevalence of inadequate intakes, and confirm their validity based on the EARs set by International Zinc Nutrition Collaborative Group. Next, the appropriateness of serum zinc as a biochemical marker for population zinc status is confirmed by a summary of: (a) the response of serum zinc concentrations to zinc intakes; (b) usefulness of serum zinc concentrations to predict functional responses to zinc interventions; (c) relationship between initial serum zinc and change in serum zinc in response to interventions. Height- or length-for-age was chosen as the best functional outcome after considering the responses of growth, infectious diseases (diarrhoea, pneumonia), and developmental outcomes in zinc supplementation trials and correlation studies. The potential of other zinc biomarkers such as zinc concentrations in hair, cells, zinc-metalloenzymes, and zinc-binding proteins, such as metallothionein, is also discussed. Molecular techniques employing reverse transcriptase (RT)-polymerase chain reaction to measure mRNA in metallothionein and ZIP1 transporter hold promise, as do kinetic markers such as exchangeable zinc pools (EZP) and plasma zinc turnover rates. More research is needed to establish the validity, specificity, sensitivity, and feasibility of these new biomarkers, especially in community-settings.

Magnesium (Mg) is the fourth most abundant mineral in the body and the most abundant intracellular divalent cation, with essential roles in many physiological functions. Consequently, the assessment of Mg status is important for the study of diseases associated with chronic deficiency. In spite of intense research activities there is still no simple, rapid, and accurate laboratory test to determine total body Mg status in humans. However, serum Mg < 0·75 mmol/l is a useful measurement for severe deficiency, and for values between 0·75 and 0·85 mmol/l a loading test can identify deficient subjects. The loading test seems to be the gold standard for Mg status, but is unsuitable in patients with disturbed kidney and intestinal functions when administered orally. There is also a need to reach a consensus on a standardized protocol in order to compare results obtained in different clinical units. Other cellular Mg measurements, such as total or ionized Mg, frequently disagree and more research and systematic evaluations are needed. Muscle Mg appears to be a good marker, but biopsies limit its usefulness, as is the case with bone Mg, the most important but heterogeneous Mg compartment. The development of new and non invasive techniques such as nuclear magnetic resonance (NMR) may provide valuable tools for routinely analysing ionized Mg in tissues. With the development of molecular genetics techniques, the recent discovery of Transient Receptor Potential Melastatin channels offers new possibilities for the sensitive and rapid evaluation of Mg status in humans.

Human selenium (Se) requirements are currently based on biochemical markers of Se status. In rats, tissue glutathione peroxidase-1 (Gpx1) mRNA levels can be used effectively to determine Se requirements; blood Gpx1 mRNA levels decrease in Se-deficient rats, so molecular biology-based markers have potential for human nutrition assessment. To study the efficacy of molecular biology markers for assessing Se status in humans, we conducted a longitudinal study on 39 subjects (age 45 ± 11) in Reading, UK. Diet diaries (5 day) and blood were obtained from each subject at 2, 8, 17 and 23 weeks, and plasma Se, glutathione peroxidase (Gpx3) enzyme activity, and selenoprotein mRNA levels were determined. There were no significant longitudinal effects on Se biomarkers. Se intake averaged 48 ± 14 μg/d. Plasma Se concentrations averaged 1·13 ± 0·16 μmol/l. Plasma Se v. energy-corrected Se intake (ng Se/kJ/d) was significantly correlated, but neither Gpx3 activity v. Se intake (ng Se/kJ/d) nor Gpx3 activity v. plasma Se was significantly correlated. Collectively, this indicates that subjects were on the plateaus of the response curves. Selenoprotein mRNAs were quantitated in total RNA isolated from whole blood, but mRNA levels for Gpx1, selenoprotein H, and selenoprotein W (all highly regulated by Se in rodents), as well selenoprotein P, Gpx3, and phospholipid hydroperoxide glutathione peroxidase were also not significantly correlated with plasma Se. Thus selenoprotein molecular biomarkers, as well as traditional biochemical markers, are unable to further distinguish differences in Se status in these Se replete subjects. The efficacy of molecular biomarkers to detect Se deficiency needs to be tested in Se-deficient populations.

Folate and the metabolically related B-vitamins, vitamin B12 and riboflavin, have attracted much scientific and public health interest in recent years. Apart from a well established role in preventing neural tube defects (NTDs), evidence is emerging to support other potential roles for folate and/or related B-vitamins in protecting against cardiovascular disease (especially stroke), certain cancers, cognitive impairment and osteoporosis. However, typical folate intakes are sub-optimal, in that although adequate in preventing clinical folate deficiency (i.e. megaloblastic anaemia) in most people, they are generally insufficient to achieve a folate status associated with the lowest risk of NTDs. Natural food folates have a limited ability to enhance folate status as a result of their poor stability under typical cooking conditions and incomplete bioavailability when compared with the synthetic vitamin, folic acid (as found in supplements and fortified foods). Current folate recommendations to prevent NTDs (based primarily on folic acid supplementation) have been found to be ineffective in several European countries. In contrast, in North America and Chile, the policy of mandatory folic acid-fortification has proven itself in terms of lowering the prevalence of NTD, but remains controversial because of concerns regarding potential risks of chronic exposure to high-dose folic acid. In the case of vitamin B12, the achievement of an optimal status is particularly difficult for many older people because of the common problem of food-bound B12 malabsorption. Finally, there is evidence that riboflavin status is generally low in the UK population, and particularly so in younger women; this warrants further investigation.

Dietary Reference Intake (DRI) values exist for vitamins and minerals, and provide a guideline on the optimal dose range to avoid deficiency and prevent toxicity. Polyphenols are widely distributed in plant foods, and have been linked to improved human health through reduced risk of chronic diseases, especially cardiovascular. Although they do not cause classical deficiencies, recently they have been discussed as ‘lifespan essentials because they are needed to achieve a full lifespan by reducing the risk of a range of chronic diseases. A recent meta analysis shows promising actions of polyphenols from cocoa, soya and tea on flow mediated dilation, blood pressure and LDL cholesterol. Many epidemiological studies support the action of polyphenols or polyphenol-rich foods on health, but there are still many gaps in our knowledge. More adequately powered, randomised, placebo controlled human studies are needed on polyphenols. There is a large number of structurally different polyphenols which are relevant for health, and obtaining enough information to set a DRI for each of these will not be feasible in the foreseeable future. A new approach is needed, and a new way of thinking, which would apply not only to polyphenols but also to other phytochemicals. Today, a target intake value of polyphenols as ‘lifespan essentials’ needs to be based on the amount of polyphenols in ‘5-a-day’. We are heading in the right direction towards a DRI, but bioavailability and dose-effects, including toxic levels, need to be established before DRIs can be considered.

This review examines the extent to which transcriptomic methods have lived up to their promise in the context of nutrition research, placing particular emphasis on examples from micronutrient research. A case is made that the high quality platform technologies now available, together with established standards and systems for data storage and exchange and powerful new methods of data analysis, mean that microarrays have reached a level of technical maturity at which they can be exploited to their full potential. In the context of nutrition and micronutrient research, transcriptomic methods have already been widely applied, albeit primarily in studies using cell lines and animal models. Using this type of approach, a multitude of genes regulated at the mRNA level by dietary components has been identified and this, in turn, has provided new insights into the biological processes affected by nutritional parameters. Evidence from the very limited number of published transcriptomics-based nutritional studies performed in human volunteers suggests that, with appropriate study design, it is feasible to apply transcriptomic methods successfully in dietary intervention trials. On the other hand, gene expression-based biomarker development still poses a major challenge. Here the use of expression profile ‘signatures’, rather than single genes, may provide a solution. Approaches designed to identify such ‘signatures’ are being developed and tested widely, primarily in the context of medical research. The applicability and power of such approaches should also be evaluated in the context of nutrition.

Nutrition research has slowly started to adopt the proteomics techniques to measure changes in the protein complement of a biological system. This enables modelling of biological processes in response to dietary interventions, as well as the elucidation of novel biomarkers for health or disease that are sensitive to such interventions. There are limited studies on the effect of micronutrients on the proteome, so this review concentrates rather more on dietary intervention studies that have used proteomics (mainly classical 2D gel electrophoresis combined with mass spectrometry) to elucidate changes in pathways that relate to glucose and fatty acid metabolism, oxidative stress, anti-oxidant defence mechanisms and redox status. The ability to measure regulation of more low abundant proteins, such as those involved in inflammatory pathways, as well as the evaluation and validation of newly discovered candidate biomarkers in human biofluids, may depend on the introduction of more quantitative and sensitive methods like multiple reaction monitoring (MRM) and multiplexed immunoassays in nutrition research.

Micronutrients are involved in specific biochemical pathways and have dedicated functions in the body, but they are also interconnected in complex metabolic networks, such as oxidative-reductive and inflammatory pathways and hormonal regulation, in which the overarching function is to optimise health. Post-genomic technologies, in particular metabolomics and proteomics, both of which are appropriate for plasma samples, provide a new opportunity to study the metabolic effects of micronutrients in relation to optimal health. The study of micronutrient-related health status requires a combination of data on markers of dietary exposure, markers of target function and biological response, health status metabolites, and disease parameters. When these nutrient-centred and physiology/health-centred parameters are combined and studied using a systems biology approach with bioinformatics and multivariate statistical tools, it should be possible to generate a micronutrient phenotype database. From this we can explore external factors that define the phenotype, such as lifestage and lifestyle, and the impact of genotype, and the results can also be used to define micronutrient requirements and provide dietary advice. New mechanistic insights have already been developed using biological network models, for example genes and protein-protein interactions in the aetiology of type 2 diabetes mellitus. It is hoped that the challenge of applying this approach to micronutrients will, in time, result in a change from micronutrient oriented to a health oriented views and provide a more holistic understanding of the role played by multiple micronutrients in the maintenance of homeostasis and prevention of chronic disease, for example through their involvement in oxidation and inflammation.