Final evidence report as part of preparatory work for the setting of Dietary Reference Values for magnesium, copper and phosphorus
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.
In 2005, the European Food Safety Authority (EFSA) received a mandate from the European Commission to review the existing advice of the Scientific Committee for Food (SCF) published in 1993 on Dietary Reference Values (DRVs) for energy, macro- and micronutrients and other substances with a nutritional or physiological effect. This report summaries current general scientific information for magnesium, copper, and phosphorus.
The data was collated via a review of scientific literature using a pre-defined search strategy which defined the literature databases (Google Scholar and PubMed) to be used and the search strings applied. This strategy also set up the boundaries for the review. Data collated would be for healthy adults (18-70 year old) and included scientific peer reviewed literature published in the English language only. Details of each document (bibliography, short abstract, categorisation tags, search details – date/time, database & search terms) were logged onto MS Excel spreadsheets and these were imported into a searchable MS Access database using bespoke software such that duplicates were removed and a number of data checks were performed. This database and software was then used to generate the reference list for the report and an EndNote™ library. A total of 183 documents for magnesium, 181 documents for copper and 167 documents for phosphorus were used in the review production. The findings of review for magnesium, copper and phosphorus are summarised in the following three sections.
Magnesium is a light, solid metallic element in the second group (alkaline earth metal) of the periodic table. It is very abundant in the natural environment and is found in many different mineral deposits. However, the most plentiful resource is the hydrosphere where magnesium exists in its ionic form (Mg2+). Magnesium has the atomic number 12 and an atomic mass of 24.312 Da. It has a brilliant, silver-white colour with a hexagonal close-packed crystalline structure and it is quite malleable. It is divalent and strongly reactive, reacting with water, many different acids and compounds such as carbon monoxide, carbon dioxide, sulphur dioxide and nitric oxide.
It is generally accepted that assessment of magnesium status in humans is problematic mainly due to the extensive distribution of magnesium inside cells and bones. Nevertheless there are three main approaches to the analysis of clinical samples: analysis of serum/plasma, metabolic studies using the Magnesium Loading Test and analysis of free magnesium in human cells and organelles. Historically, several different analytical methods have been used for quantifying magnesium in clinical samples. These include atomic absorption spectroscopy, atomic emission spectrometry, colorimetry, fluorometry, compleximetry, and chromatography. For free magnesium, ion-specific microelectrodes and fluorescent probes are a common approach and Nuclear Magnetic Resonance (NMR) spectroscopy can also be used where the equipment is available.
Magnesium is an essential mineral for a number of metabolic activities, particularly those associated with cellular function. It is estimated that more than 300 enzymatic reactions require magnesium and these include metabolism of proteins, lipids, carbohydrates, nucleic acids, synthesis of H2 transporters and all reactions involving ATP. The mechanism for magnesium involvement includes chelation to form complexes, e.g. MgATP complex, which are an active substrate for enzyme activity or direct binding to a protein resulting in allosteric changes.
There have been numerous studies over the last few decades that have linked magnesium deficiency and a wide range of different health conditions and as a consequence there has been a significant growth in interest from health professionals regarding the role magnesium plays in health and disease.
The clinical manifestation of magnesium deficiency caused by dietary deprivation in otherwise healthy populations is variable and may include neuromuscular hyperexcitability, cardiac arrhythmias, increased muscular tension and cramps, increased susceptibility to stress, headaches, irritability and reduced ability to concentrate. A deficiency may also lead to a disturbance of the normal body distribution of trace metals. Severe deficiency can lead to hypocalcemia.
Magnesium deficiency has also been linked with a range of chronic diseases including hypertension and cardiovascular diseases, cancer, type 2 diabetes mellitus, Alzheimer's disease, migraine headaches, clinical depression, osteoporosis and poor bone health. It is also believed that magnesium deficiency may be a factor in pre-eclampsia and eclampsia, various neuromuscular conditions, pre-menstrual syndrome, and thyroid disease.
The human body contains approximately 1 mol of magnesium, i.e. 20-30 g, approximately distributed as follows: 0.3% serum, 0.5% red blood cells, 19% soft tissue, 27% muscle and 53% bone).
Serum magnesium is a poor biomarker of intake, responding only slowly to (overt) changes in intake. Serum, plasma, and urine magnesium concentrations have been suggested as biomarkers of magnesium status in the general population. However, assessment of magnesium status via these markers is problematic as the majority of body magnesium is intracellular and within bones. Analysis of magnesium concentration in red blood cells (to assess intracellular magnesium) is reported as not correlating well with total body magnesium and is not considered to be a reliable indicator of magnesium status.
Knowledge of the genetic regulation of magnesium status is limited, yet genetic factors contribute to the control of magnesium status. It has been suggested that about 27% of the variance in serum magnesium concentration is genetically determined.
Copper is a solid chemical element belonging to group II of the periodic table. It occurs at approximately 0.006% in the Earth's crust and small amounts are also found in fresh and marine waters. Metallic deposits are rare but copper-minerals are found throughout the world. Copper has the atomic number 29 and an atomic mass of 63.55 Da. It has a reddish-gold colour with a face-centred cubic crystalline structure. Copper is malleable and an excellent conductor of both electricity and heat. Its chemistry is dominated by the +2 oxidation state but there is also substantial chemistry for the +1 state. It readily forms inorganic and organometallic compounds.
The human body contains approximately 0.02 to 0.03 mmol (1.4 to 2.1 mg) of copper per kg body weight which is typically up to around 0.79 to 2.36 mmol (50 to150 mg) of copper in total. Around half of this is found in muscle with significant amounts in the bones and liver. Copper concentrations in tissues range from 1 to 2 μg g-1, except in kidney, liver, brain, heart and skeleton where the range is between 4.8 to 12 μg g-1. There is very little free copper in the human body. The vast majority (95%) is in blood plasma bound in a protein complex, ceruloplasmin and the remaining 5% is loosely bound to albumin.
The main dietary sources of copper are crustaceans and shellfish, whole grains, beans, seeds, nuts especially pecans, cashews and walnuts, dried fruits, dark leafy greens such as spinach, potatoes, mushrooms and organ meats.
There are a wide range of techniques available for analysis of copper in clinical samples including atomic absorption spectrometry (AAS) with flame detection, graphite furnace atomic absorption spectroscopy, inductively coupled plasma atomic emission spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and stabilized temperature platform graphite furnace atomic absorption. Atomic absorption spectroscopy is the most common approach for measurement of copper in foodstuffs.
Copper is an essential micronutrient that has a number of biological functions. It is found in practically every cell of the human body. Virtually all organisms, including man, require copper as a catalytic cofactor for biological processes including respiration, iron transport and absorption, circulation, oxidative stress protection, peptide hormone production, pigmentation, blood clotting and normal cell growth and development. Copper is involved in the function of several enzymes, including cytochrome-c oxidase, lysyl oxidase, dopamine hydroxylase, amino acid oxidase, superoxide dismutase and monoamine oxidase.
Dysfunction of copper metabolism leading to deficiency or excess can lead to serious disease. Dysfunction of copper metabolism caused by genetic abnormalities serves as an illustration of the importance of copper for good health as shown by two well documented disorders: Menke's disease (which presents as copper deficiency) and Wilson's disease (presenting as copper toxicity).
Copper deficiency is rare in healthy populations but is more likely to occur early in life, especially in premature infants. It has also been reported in patients with malabsorption syndromes such as celiac disease and cystic fibrosis. When symptomatic, copper deficiency can manifest as bone abnormalities, anaemia and neutropenia. Less frequent signs and symptoms of copper deficiency include hypopigmentation of the hair, hypotonia, increased susceptibility to infection, and abnormalities in metabolism of glucose.
There are also a number of chronic adverse health effects linked with copper deficiency including impaired oxidative defence, poor prostaglandin metabolism and cardiovascular disease. Human and animal studies have also shown that copper deficiency could be a factor in rheumatoid arthritis.
The small intestine is the most important site for absorption of copper from the gastrointestinal tract. The proportion of copper absorbed by the gastrointestinal tract decreases with increasing dietary copper intake, however the absolute amount of copper absorbed increases. Copper is primarily eliminated through bile and faeces with bile playing an important role in maintaining copper homeostasis. Urine is a minor route for copper elimination and this route does not contribute to copper homeostasis regulation.
Serum copper is an unreliable marker of intake other than in cases of low intake. Plasma and erythrocyte markers are more reliable for diagnosing a deficient intake of copper, however, their utility in assessing excess intake is less reliable as copper toxicity is not associated with increased serum copper of ceruloplasmin.
Serum copper or ceruloplasmin are the predominant biomarkers to determine copper status, however, their poor specificity and sensitivity undermine their utility in this role. Nevertheless, serum copper is a reliable marker in cases of severe deficiency and is considered useful at the population level. Erythrocyte superoxide dismutase is sensitive to changes in copper status; however, there is no agreed standard assay method, which limits its value as a biomarker.
Much of the knowledge of typical copper homeostastis is derived from understanding the molecular basis of genetic diseases in copper metabolism, namely, Menke's disease and Wilson's disease, which manifest as a result of mutations in copper-transporting membrane ATPases.
Phosphorus is a non-metallic solid chemical element, found in the pnictogen group of the period table. It is very abundant in the natural environment and is the 11th most common element. Phosphorus does not occur in nature as a free element due to its high reactivity but is found in the form of phosphate minerals. It has the atomic number 15 and an atomic mass of 30.974 Da. Pure phosphorus exists as several allotropes, the most common being the white (sometimes yellow), red and black forms. Less common forms include violet and scarlet phosphorus, and the gaseous form diphosphorus (P2). White phosphorus is a waxy, transparent crystalline solid with a faint garlic-like odour. It sometimes appears slightly yellowish because of traces of red phosphorus. It is this allotrope that occurs most commonly at room temperature. Red phosphorus is a red powder, odourless and is stable in air. Black phosphorus looks like graphite powder.
There are many methods for the assessment of phosphorus in body fluids. Most are based on the colorimetric method and depend on the development of the phosphomolybdate complex in the presence of a reducing agent to form a distinctive blue colour. Other methods include a combined enzyme-substrate system and redox electrodes.
Phosphorus is vitally important in a host of biological functions. A delicate balance is required between the body's phosphorus and calcium loads, without this balance various biological functions may be disrupted. Phosphorus, alongside calcium, is a principal component of bones and teeth. Phosphorus also interacts with vitamin D, consequently, phosphorus has a role in the performance of the endocrine system. Phosphorus is also a critical component of cell structure – it combines with lipids to form various types of phospholipids, a major structural component of all cell membranes throughout the body. Another important role of phosphorus is that of energy production and chemical storage. All energy production and storage processes are dependent on phosphorylated compounds such as adenosine triphosphate (ATP), adenosine diphosphate (ADP) and creatine phosphate. Other functions include: a component of nucleic acids; a buffering agent, maintaining the pH balance in order to maintain osmotic pressure; and the phosphorus-containing molecule 2,3-diphosphoglycerate binds to hemoglobin in red blood cells and affects oxygen delivery to the tissues of the body.
Phosphorus is ingested from food in a variety of forms. It is found in most foods, with the richest sources being the protein-rich food groups such as dairy, eggs, grains, nuts, meat and fish. However, current food processing practices include significant use of phosphate additives. Consequently, processed foods have high phosphate content. Some foods contain phosphorus compounds which have been associated with adverse health effects. For example, carbonated beverages often contain phosphoric acid, which may contribute to erosion of tooth enamel and has also been associated with stomach and intestinal irritation, and urinary changes that can promote kidney stones, as well as bone resorption. Phytate, the principal storage form of phosphorus in many plant tissues, appears to potentially have both beneficial and adverse health effects. Phytates forms complexes with dietary minerals, especially iron and zinc, which may cause mineral-related deficiency. However, there is also evidence that phytates function as an antioxidant in the human body.
Phosphorus deficiency has been linked with the risk of hypophosphatemia; myocardial dysfunction due to hypophosphatemia; and rhabdomyolysis, or acute necrosis of skeletal muscle tissue. However, phosphorus deficiency is rare in the general population.
Hyperphosphatemia, an electrolyte disturbance in which there is an abnormally elevated level of serum phosphate, may be caused by increased phosphate loading due to endogenous or exogenous sources such that the body has insufficient ability to remove the excess – often due to renal failure. Clinical manifestation of hyperphosphatemia includes tetany and seizures due to hypocalcemia. Concerns have been raised over the role of dietary phosphorus in bone health and in particular the significance of the dietary ratio of calcium to phosphorus (P), specifically diets that contain a higher proportion of phosphorus to calcium (mass ratio of Ca:P ≤0.5).
Total body phosphorus in adult man is typically in the order of 400-600g or <1% of body weight and approximately 85% of this is located in the bones and teeth. Although this can be considered a medium term reservoir of phosphorus, it is not a static body store as approximately 300 mg of bone phosphorus is resorbed and reformed daily. The rate of turnover is increased by dietary intake with a low ratio of calcium to phosphorus and is also influenced by protein intake. Cells hold very limited reserves of inorganic phosphate relying on supply by extracellular fluid. Adults' total phosphorus intake is approximately 1500 mg/day and due to effective absorptive mechanisms the efficacy of phosphorus absorption can reach approximately 60-70%. The absorbed phosphorus enters the exchangeable phosphorus pool which mainly consists of the intracellular phosphorus and the phosphorus arising from bone mineralization. Both the intestine and kidney are involved in phosphate homeostasis by serving as regulators of phosphorus absorption from the diet and phosphorus excretion, respectively. Under normal conditions the amount of phosphorus excreted in the urine is equivalent to the amount of phosphorus absorbed in the intestine. Sweat is not an important source of phosphorus loss.
Urinary biomarkers are reliable indicators of phosphorus intake. Serum or plasma concentrations of phosphorus are used to assess phosphorus status. However, this marker does have limitations and there is a need to develop more appropriate biomarkers. There is a positive correlation between phosphorus intake and serum phosphorus concentration at lower dietary phosphorus intake, however, at higher intake this relationship tends to weaken. Dietary intake can be determined using 24-hour urine collection and measuring urinary phosphorus concentration which is considered a reliable estimate of dietary intake providing that the individual is in phosphorus balance.
Knowledge of the genetic regulation of phosphorus status and the understanding of phosphorus homeostasis is largely due to molecular studies of human genetic disorders. Cloning of the genes leading to those disorders has shown that the osteo-renal metabolic axis plays a large role in phosphorus homeostasis.