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5 Measuring intake of nutrients and their effects: the case of copper L. B. McAnena and J. M. O’Connor, University of Ulster 5.1 Introduction In this chapter, copper is considered as a case study for the measurement of the effect of nutrient intake. The importance of the role of copper in biological systems is first explored in a brief review of selected human cuproenzymes. Worldwide estimates of dietary copper requirements, and dietary recommenda- tions, are discussed. Although dietary sources of copper are numerous, many Western diets appear to be barely adequate in copper. While clinical copper defi- ciency is rare, usually seen only in malnourished children and premature babies or as a consequence of malabsorption, a proposed link between copper deficiency and degenerative diseases makes the question of suboptimal status an important issue. Copper toxicity, acute or chronic, is also rare, but sound limits for total intake and for levels of copper in drinking water are essential nonetheless. The assessment of nutrient intake, in general, is made difficult by the limitations asso- ciated with the available methods. Putative or traditional indicators of copper status are also subject to problems and limitations, and rarely fulfil all of the essential criteria for a good index of copper status. Functional copper status is the product of the interactions of copper with a variety of factors. Foods vary in copper content and digestibility, and the mechanisms involved in absorption are affected by a variety of luminal and systemic factors. Distribution of copper around the body occurs in two phases: transport from the intestine to the liver; and subsequent delivery to other tissues. Problems specific to the assessment of copper absorption are discussed. Some recent advances in copper metabolism research are outlined, along with promising new areas for future study. 5.2 The nutritional role of copper Copper was identified as an essential trace element, first for animals 1 and sub- sequently for humans 2 when anaemia was successfully treated by supplementing the diet with a source of copper. Since then the full significance of its role in bio- logical systems has continued to unfold as it has been identified in a large number of vital metalloproteins, as an allosteric component and as a cofactor for catalytic activity. These proteins perform numerous important roles in the body, relating to the maintenance of immune function, neural function, bone health, arterial compliance, haemostasis, and protection against oxidative and inflammatory damage. However, the accurate assessment of copper status is problematic. Func- tional copper status is the product of many interacting dietary and lifestyle factors, and an adequate marker of body copper status has yet to be identified. Accurate measurement of dietary copper intake is difficult because while a number of dietary factors are known to limit copper bioavailability, the precise molecular mechanisms of copper absorption and metabolism are not completely understood. Shown in Table 5.1 is a selection of the copper-containing enzymes and pro- teins known to be important in human systems. A number of these enzymes exhibit oxidative/reductive activity and use molecular oxygen as a co-substrate. In these redox reactions, the ability of copper to cycle between cupric and cuprous states is crucial to its role as electron transfer intermediate. Cytochrome-c 118 The nutrition handbook for food processors Table 5.1 Human copper-containing proteins, and their functions Protein Function Cytochrome-c oxidase Cellular energy production Ferroxidase I (Caeruloplasmin) Iron oxidation and transport; free radical scavenging; amine and phenol oxidation; acute-phase immune response Ferroxidase II Iron oxidation Hephaestin Iron metabolism Copper/zinc superoxide dismutase Antioxidant defence Extracellular superoxide dismutase Antioxidant defence Monoamine oxidase Brain chemistry Dopamine betatwo-hydroxylase Brain chemistry Diamine oxidase Limitation of cell growth, histamine deactivation Lysyl oxidase Connective tissue formation Peptidylglycine a-amidating Peptide hormone activation monooxygenase Prion protein PrP Antioxidant defence and/or copper sequestration and transport Tyrosinase Melanin synthesis Albumin Metal binding in plasma and interstitial fluids Chaperone proteins Intracellular copper delivery to specific target proteins Chromatin scaffold proteins Structural integrity of nuclear material Clotting factors V and VIII Thrombogenesis Metallothionein Metal sequestration Transcuprein Copper binding in plasma oxidase, embedded in the inner mitochondrial membrane, is the terminal link in the electron transport chain. It catalyses the reduction of oxygen to water. One molecule of cytochrome-c oxidase contains three copper atoms and possesses two active sites. At one site two copper atoms receive, from the electron-carrier cytochrome-c, electrons which are then transferred to the second active site, where the third copper atom functions as a reducing agent. 3 Because this is the rate-limiting step in electron transport, cytochrome-c oxidase is considered the single most important enzyme of the mammalian cell. Ferroxidases I and II are plasma glycoproteins. Ferroxidase I, also known as caeruloplasmin, oxidises Fe (II) to Fe (III) without formation of hydrogen per- oxide (H 2 O 2 ) or oxygen radicals. It is primarily this role which gives rise to caeru- loplasmin’s well-known antioxidant function. It also scavenges H 2 O 2 , superoxide and hydroxyl radicals, and inhibits lipid peroxidation and DNA degradation stimulated by free iron and copper ions. 4 Caeruloplasmin is also an acute-phase protein: in acute response to inflammatory cues caeruloplasmin concentration rises, binding free circulating iron and limiting the amount available to partici- pate in oxidative reactions. One molecule of caeruloplasmin contains six copper ions, of which three provide active sites for electron transfer processes, while the remaining three together form an oxygen-activating site for the enzyme’s catalytic action. 5 Superoxide dismutase (SOD) is another important and well- studied enzyme. In human systems, it exists in several forms, of which two contain copper: the cytosolic copper/zinc variety sometimes termed SOD1, present in most cells; and the extracellular SOD2, found in the plasma and also in certain cell types in the lung, thyroid and uterus. 6 SOD catalyses the dismuta- tion of superoxide radicals to hydrogen peroxide and oxygen. In several amine oxidases, copper acts as an allosteric component, conferring the structure required for catalytic activity. Monoamine oxidase (MAO) inacti- vates, by deamination, substrates such as serotonin and catecholamines includ- ing adrenalin, noradrenalin and dopamine. Tricyclic antidepressants are MOA inhibitors. Diamine oxidase (DAO) deaminates histamine and polyamines involved in cell proliferation. It is present at low levels in the plasma, but at higher concentrations in the small intestine where histamine stimulates acid secretion, in the kidney where it likely inactivates diamines filtered from the blood, and in the placenta, where it is thought to inactivate foetal amines in maternal blood. Lysyl oxidase deaminates lysine and hydroxylysine, which are present as sidechains of immature collagen and elastin molecules. It thereby enables the formation of crosslinks which lend strength and flexibility to mature connective tissue. Peptidyl-glycine a-amidating mono-oxygenase (PAM) is found in the plasma and in a number of tissues, including the brain. It produces mature, a-amidated, peptide hormones from their glycine-extended precursors. The enzyme contains two copper atoms per molecule. 7 Dopamine b-hydroxylase (DbM) is a mono- oxygenase similar to PAM in structure and activity. Found in the adrenal gland and the brain, it catalyses the synthesis of the catecholamines adrenalin and noradrenalin from dopamine. Tyrosinase, or catechol oxidase, is the only enzyme involved in the synthesis of melanin from tyrosine. Tyrosinase first hydroxylates the amino acid to dopa, then oxidises it to dopaquinone. Subsequent reactions Measuring intake of nutrients and their effects: the case of copper 119 leading to melanins occur spontaneously in vitro. Regulation of pigment forma- tion is also provided by tyrosinase, as it can remove substrates from this pathway by catalysing alternative reactions for them. 8 Congenital deficiency of tyrosinase results in albinism. In the nucleus, copper has a structural role as an essential component of chromatin scaffold proteins, which contribute to nuclear stability. 9,10 It does not, however, appear to be required for DNA synthesis in mammalian cells. Although in yeast cells, copper has been identified as a component of gene regulatory mech- anisms, if equivalent proteins exist in human cells they remain to be identified. 11 5.3 Dietary copper requirements Despite the known essentiality of copper in humans, dietary requirements are still uncertain. World-wide, a number of Dietary Reference Values are recommended for copper intake (see Table 5.2) but the variability between them is indicative of the lack of consensus between advisory bodies. Making dietary recommenda- tions, even of Estimated Average Requirements (EAR), is difficult owing to a lack of adequate data. In the UK, the Department of Health considers the avail- able data on human copper requirements to be insufficient to determine an EAR. 12 In the US, an EAR of adults for copper was derived from a combination of bio- chemical indicators of copper requirement, as no single indicator was judged as sufficiently sensitive, specific and consistent to be used alone. A Recommended Daily Allowance (RDA) can be calculated by extrapolating the EAR to account for inter-individual variation in requirements. The US RDA, like the UK Reference Nutrient Intake (RNI) is intended to provide enough copper for about 97% of adults. The World Health Organization has loosely defined an Acceptable Range of Oral Intake (AROI). Its upper limit could not be specifically confirmed because of the limited information available on the level of intake that would provoke adverse heath effects. It is apparent that more data are needed if sound and defensible guidelines are to be derived. 5.4 Sources of copper In most diets, sources of copper are numerous because copper is widespread in foods. Rich sources include organ meats, nuts, shellfish, seeds, legumes and the germ portion of grains. Other foods including cereals, meats, mushrooms, pota- 120 The nutrition handbook for food processors Table 5.2 Dietary Reference Values for copper Dietary Reference Value Copper (mg/d) Source US EAR 0.7 Food and Nutrition Board, 2001 US RDA 0.9 Food and Nutrition Board, 2001 UK RNI 1.2 Department of Health, 1991 WHO AROI 1.2 to 2 or 3 WHO International Programme on Chemical Safety, 1998 toes, tomatoes, bananas and other dried fruits provide sufficient copper in a normal diet to ensure that overt copper deficiency is rare in human populations. Nonethe- less, many Western diets are estimated to supply a level of copper only barely adequate to meet the body’s requirements. Published estimates of copper intake vary around 1–2 mg/d, with few diets containing more than 2 mg/d. 13,14,15,16,17 5.5 Copper deficiency Clinical copper deficiency is seen mainly in malnourished and recovering chil- dren, in premature babies, in patients receiving total parenteral nutrition (TPN) and as a consequence of malabsorption. Copper deficiency also occurs as the result of Menkes syndrome, a rare inherited defect of copper transport. Mal- nourished children are reported to be at particular risk of copper deficiency. A diet consisting exclusively or predominantly of cow’s milk, with its poor bioavail- ability of copper, increases the likelihood of copper malabsorption. During nutri- tional recovery, growth rate can be 5–10 times the normal rate, increasing copper requirements beyond the dietary intake. 3 Copper deficiency during this period has been shown to impair growth rate 18 and to be associated with increased incidence of respiratory infection. 19 Preterm babies are also at particular risk of copper deficiency, for several reasons. Copper stores are acquired late in foetal development, as metallothionein- bound copper accumulates in the foetal hepatocyte nuclei over the last trimester. 11 Although neonates appear not to absorb copper well, particularly from highly- refined carbohydrate-based diets or cow’s milk 20 , full-term infants have well- developed copper stores which can be mobilised during the first six months’ rapid growth, to supplement dietary intake. 21 Full-term infants are therefore independent of dietary intake for the first weeks of life. 22 Premature babies, especially those with very low birth-weight, do not have such a resource. They also have higher growth rate than full-term babies, with accordingly higher copper requirements. 23 Clinical copper deficiency in adults was unknown until the introduction of TPN, which is now well known to result in elevated urinary copper output and a net depletion of copper status. 20 Although copper is now usually added to TPN infusates, it is often withheld from cholestatic patients since their impaired biliary excretion is expected to result in reduced intestinal losses. The complex interac- tions between disease states and copper metabolism, however, make individuals’ requirements difficult to anticipate, and TPN-related copper deficiency continues to occur. 24,25 Anumber of malabsorption syndromes have been reported to result in increased intestinal copper losses leading to deficiency. Such conditions include coeliac disease 26 , cystic fibrosis 27 , shortened intestine following surgery 28 , and chronic or recurrent diarrhoea. 29,30 Menkes disease is an X-linked recessive disorder of copper metabolism in which mutations in the MNK gene impair copper transport from cells. The disease is manifest as copper deficiency, because although copper is absorbed by gut cells, very little is transported to the tissues where it is required Measuring intake of nutrients and their effects: the case of copper 121 for enzyme function. Symptoms usually appear within the first months of life, and can result in death in early childhood. 31 In clinical copper deficiency, the most common defects are: cardiovascular and haematological disorders including iron-resistant anaemia, neutropenia and thrombocytopenia; bone abnormalities including osteoporosis and fractures; and alterations to skin and hair texture and pigmentation. 23 Immunological changes have also been indicated. 19,32 These changes may be accompanied by depressed serum copper and blood cupro- enzymes, with caeruloplasmin concentrations observed at 30% of normal. 6 It has been clearly demonstrated that very many of the changes induced by severe copper deficiency are also risk factors for ischaemic heart disease in humans. Human copper depletion studies have produced impaired glucose clearance, 33 blood pressure changes, 34 electrocardiographic irregularities and significantly increased LDL cholesterol with decreased HDL cholesterol. 21 In copper-deficient animals, cardiovascular disorders observed include lesion and rupture of blood vessels, cardiac enlargement, myocardial degeneration and infarction (MI). 33 It has been argued that copper deficiency is the only nutritional deficit known to affect adversely so many risk factors for ischaemic heart disease. 35 The proposed link between copper deficiency and cardiovascular disease is supported by data gathered from studies of cardiovascular patients. Post-mortem measurement of tissue copper has revealed lower-than-normal copper concentrations in ischaemic hearts, in the liver and heart of individuals with severe atherosclerosis, and in leucocytes of patients with highly occluded coronary arteries. 33 A variety of mechanisms may contribute to the cardiovascular effects of copper deficiency. There is evidence for alterations in the activity of copper- dependent enzymes, increased oxidative stress and damage to biomolecules, and interference with the maintenance of blood pressure. An interaction of these three mechanisms of damage has been proposed to have even further potential for harm, 36 which need not be limited to cardiovascular defects. The adverse effects elicited by copper deficiency are numerous and as varied as the roles of copper in health. In the light of this, it has been proposed that long-term sub-clinical copper deficiency may contribute to the pathogenesis of a number of degenera- tive and inflammatory conditions. 37 5.6 Copper toxicity Copper toxicity is rare because levels in food and water are generally low and because increased dietary intake results in decreased absorption and increased excretion. 38 Cases of both acute and chronic poisoning have, however, been reported. Acute toxicity has been known to result from accidental or deliberate consumption of copper salts and, more commonly, from contamination of drinks by copper containers. 6 A 1957 report of contamination of cocktails stored for just two hours in a metal cocktail shaker was used in 1988 by US Environmental Pro- tection Agency (EPA) Office of Drinking Water to derive drinking water regula- 122 The nutrition handbook for food processors tions which are still in place. 39 Acute toxicity results, initially, in symptoms such as abdominal pain, nausea, vomiting and diarrhoea. These gastrointestinal effects are often sufficiently severe and prompt to prevent systemic toxicity which, like chronic copper poisoning, is associated with liver damage. Chronic toxicity has most often been caused by contaminated water supplies, and occasionally by contamination of haemodialysis equipment by copper parts. 40 The highest intake which has been shown experimentally to produce no adverse effects is defined as the No-Observed-Adverse-Effects-Level (NOAEL). While a NOAEL of 4 mg/l in drinking water has been observed for acute effects, 41 a higher NOAEL of 10 mg supplemental copper per day has been demonstrated to provoke no ill effect upon liver function after 12 weeks. 42 The US Food and Nutrition Board have used the latter value to calculate a theoreti- cal Tolerable Upper Intake Level (UL), defined as the highest level of daily intake considered likely to pose no threat to the health of almost all individuals. The UL is in agreement with the World Health Organization’s provisional maximum tolerable daily intake (PTDI), an estimate of the amount that can be ingested daily over a lifetime without appreciable risk to health. In drinking water, copper levels vary considerably depending on factors including the pH and hardness of the water supply and the length of piping. In some systems, copper salts are added to control the growth of algae. 16 Suggested upper limits for copper in drinking water differ world-wide, and while some are based on health issues, others consider only aesthetic values. The issue is currently under review by several international groups. 39 Table 5.3 shows current permissible levels of copper in drinking water, and recommended limits of total copper intake. A number of disorders of copper homeostasis can result in toxicity leading to liver cirrhosis at dietary copper levels which are tolerated by the general popu- lation. Copper-induced cirrhosis is mainly restricted to children, possibly because Measuring intake of nutrients and their effects: the case of copper 123 Table 5.3 Recommended limits of copper intake Reference Value Copper limit Source In drinking water (mg/l) UK standard 3.0 Water Supply (Water Quality) Regulations, 1989 WHO standard 2.0 WHO Guidelines for Drinking Water Quality, 1993 EU standard 2.0 EU Directive 98/83 L330, 32–54 US maximum 1.3 EPA Drinking Water Regulations, 1988 contaminant level Total intake (mg/d) US UL 10.0 Food and Nutrition Board, 2001 WHO PTDI 10.0 (women) World Health Organization, 1996 12.0 (men) of the lower capacity of their biliary excretory mechanisms. 38 Indian Childhood Cirrhosis (ICC) is a fatal condition of copper metabolism which was, at one time, a major cause of infant mortality on the Indian subcontinent. ICC sufferers, usually infants aged between 6 months and 5 years, are often found to have been exposed at an early age to milk contaminated with copper from untinned brass or copper vessels. 43 High copper intake, however, is not thought to be the sole cause of the illness; both environmental and genetic components are thought to contribute. 44 Cases of a similar infantile condition have been reported in Germany and in the Tyrol, Austria. 45,46 Incidence of both ICC and Tyrolean Infantile Cirrhosis has dropped in recent years. One possible explanation is reduced use of brass vessels, while an alternative is the dilution of the responsible gene by increased population mobility and fewer consanguineous marriages. A rare inherited disorder of copper metabolism leads to Wilson’s disease, in which copper cannot be properly transported out of the liver and so accumulates to toxic levels. When the hepatocytes die, copper is released into the plasma and deposited in other tissues including the central nervous system. 47 Treatment of Wilson’s disease is aimed at removing copper from the body and preventing its reaccumulation. 5.7 General limitations in assessing nutrient intake As for any nutrient where deficiency and toxicity are issues, the reliable assess- ment of intake is paramount. The ultimate aim of defining optimal dietary intakes is hampered by difficulties in determining certain key facts, namely, individual copper intakes and status. Dietary intake can be assessed by a number of methods, involving either the recording of actual consumption (prospective) or the assess- ment by questionnaires of diet in the recent past (retrospective). At each stage in the application of any method, errors are introduced, producing as a result either a systematic bias or random deviations from the true values. Of the methods in common use, the weighed dietary record is widely accepted to be the most accu- rate, but it requires a considerable amount of co-operation from human subjects. This disadvantage may give rise to substantial bias, most likely toward under- reporting habitual dietary intakes. 48 In clinical practice the most frequently used method of dietary assessment is the diet history, which is highly dependent on accurate recall by the individual. It is possible to verify these reports, to some extent, by independent methods. Under- and over-estimation of an individual’s total food intake can be identified by measuring total energy expenditure, either directly, using the doubly-labelled water technique, or indirectly, by calculating basal metabolic rate. Another check is a comparison of the individual’s 24-hour urinary nitrogen output with the reported protein intake. The accuracy of these methods is limited either by the involvement of estimates, or by reliance on the assumption that body weight is constant. One means of assessing nutrient requirements is the metabolic balance study. The aim of a balance study is to compare the intake of a nutrient with the amount 124 The nutrition handbook for food processors leaving the body. A constant daily intake of the nutrient in question is provided throughout the study period, and collection of stools and urine are made. Crucial to the success of the investigation is the accuracy of measurement of intake and excretion. For this reason, balance studies demand careful planning and execu- tion, good facilities for food preparation, sample collection and sample storage, and good laboratory services. A major limitation is that balance studies provide little information about nutrient transport or utilisation within the body. 22 Nutrient intake can also be assessed by the use of experimental diets with different mineral intakes. The use of experimental diets to determine nutrient requirements depends on the selection and measurement of a biochemical endpoint, to serve as a marker of nutrient sufficiency. However, experimental diets must be carefully constituted to minimise the possibility that other dietary components may modify absorption of the nutrient, or even influence directly the chosen marker. 23 A limitation of this method is that it permits the estimation of the basal nutrient requirements, but not the amount needed to maintain bodily nutrient reserves. Epidemiological studies such as the US Total Diet Study 13 or the North/South Ireland Food Consumption Survey 15 are often carried out with the aim of esti- mating the fraction of the population at risk from deficiency or excess intake. Attempts are made to assess long-term average intake of populations from data gained using short-term measures of intake. Few studies have reported testing the validity of such an extrapolation, but a recent study which examined values cal- culated from up to six samples, spaced over a year, found significant temporal variability for individual subjects. 49 In addition, when the reliability of short-term (4-day) samples was estimated by comparing individual values to the aggregate value, results suggested that three short-term samples would be required to achieve a strong correlation (r = 0.9) between short- and long-term values. Tra- ditional reliance on short-term measures for estimation of long-term copper status could produce erroneous results. 5.8 Putative copper indicators Determination of copper status suffers from the lack of sensitive, reliable and easy measures for detecting marginal copper status. Copper levels in the hair, nails or saliva do not appear to reflect copper status. 50 Urinary copper is normally extremely low, and although it can decline in extreme copper deficiency, this is usually seen only after changes are seen in other copper indices. 17 In copper de- pletion and repletion studies, cuproenzyme activities have appeared relatively insensitive to change. 51 The traditional and most commonly used putative indicator of copper status is serum or plasma copper. Under normal circumstances, strong homeostatic mechanisms maintain the range between 0.64 and 1.56mg/ml. 50 Although in severe copper depletion it has been known to fall to very low levels, and to recover upon copper repletion, it does not appear to reflect dietary levels when Measuring intake of nutrients and their effects: the case of copper 125 intake is close to normal. 17 It does not increase after a meal or decrease during short-term fasting and has been shown to correspond poorly with reported dietary intakes. 52 In some studies of copper depletion, serum copper responses have been absent even in the presence of other biochemical or physiological changes. 53 Serum copper is known to be altered by a number of factors not directly related to copper status. Concentrations are low in infancy and rise to adult levels over the first 4–6 months, or longer following a low birth weight. In adult women, serum copper concentration is generally higher than in men, and is further raised during pregnancy and by oestrogen treatments. 54 There is also a normal diurnal variation with a slight peak in the morning. Plasma copper fluctuates with age, and is raised in a number of other conditions including exercise, rheumatoid arthritis, dilated cardiomyopathy and anticonvulsant chemotherapy. Between 60 and 95% of serum copper is associated with caeruloplasmin, so serum copper levels often mirror those of caeruloplasmin. 11 Normal levels of serum or plasma caeruloplasmin protein are 180–400mg/ml. 17 Like serum copper, caeruloplasmin has shown variable responses to marginal depletion. Its concentration and activity fall with severe copper deficiency and return to normal with copper repletion; but because of its role as an acute phase protein, caeruloplasmin concentration in the plasma reflects oxidative status more reliably than copper status. Copper depletion may be masked by caeruloplasmin elevated in response to exercise, infection or inflammation, liver disease, malig- nancy and MI. 55 Like serum copper, normal caeruloplasmin values also vary with age and gender, and during pregnancy. 23 Erythrocyte copper/zinc SOD concentration is normally 0.471 ± 0.067mg/g protein. 50 SOD activity has appeared in some studies to be more sensitive than caeruloplasmin to changes in copper status, while in other studies its activity has fallen with depletion but failed to respond to repletion. 56 Copper/zinc SOD activ- ity has been reported to rise in response to physical exercise. 57 As an antioxidant enzyme, SOD is likely to respond to conditions of oxidative stress. A further com- plicating factor is that SOD measured in erythrocytes is unlikely to reflect short- term changes in dietary intake owing to the 100-day lifetime of erythrocytes. Leucocyte copper has been found to decline along with other indices of copper status. 51 Platelet copper has been shown to decline with copper depletion and to recover with copper repletion. 56 However, there are not yet sufficient experi- mental data to confirm the validity of leucocyte or platelet copper as indicators of suboptimal copper status. DAO has been indicated in some studies of copper depletion as a possible marker of copper status. 58 Its measurement is currently difficult because of its extremely low levels in plasma. Furthermore, its use as an indicator may be limited because it is elevated during pregnancy, after heparin treatment and in some conditions of intestinal damage. A valid functional index must respond sensitively, specifically and predictably to changes in the dietary supply or stores of copper, and must be measurable and accessible for measurement. Validation of a candidate marker would require demonstration of a cause and effect relationship between the marker and copper status and also, ideally, between copper status and health measures. 59 126 The nutrition handbook for food processors 5.9 Functional copper status The body’s total copper content is the end result of a balance achieved between absorption and biliary excretion. In comparison to other trace elements, relatively little copper is present in the body, usually about 100 mg. The largest tissue pool of copper is in the skeleton, followed by the muscle; 60 however, the major site for storage of exchangeable copper is the liver, 61 which contains 4–6mg/g wet weight. 60 This is followed by the brain, kidney and heart. Functional copper status, however, is not dependent only on the absolute copper content of the body. Utilisation of absorbed copper is also modulated by the interactions of copper with a number of other factors, deriving from the general status and requirements of the body. Furthermore, individual organs have the potential to modulate copper status by retaining copper in response to dietary restriction. This capacity is highly organ-specific, being stronger and/or more sen- sitive in some tissues than in others. Depletion studies in animals have found plasma to possess almost no copper conservation mechanisms, whereas the heart and brain were shown to conserve most of their endogenous copper during periods of restriction. 62 Liver copper conservation mechanisms, while induced only after levels had dropped to around 60% of normal, were thereafter found to operate so strictly that almost no copper was exported into the plasma, and biliary copper excretion was also significantly reduced. 5.10 Mechanisms of copper absorption Copper absorption in humans has been found to depend on a number of factors, of which the most important is probably dietary copper intake. 6 The efficiency of copper absorption is regulated to maintain body copper status, with levels of uptake rising to 70% during periods of deficiency, 63 and falling to 12% in high-copper diets. 61 This modulation of absorption, which provides a means of adapting to changing dietary intake, appears to develop during childhood, with copper absorption in infants operating at a lower level than in adults. 38 While a low level of copper absorption occurs in the stomach, the main site of absorption is the duodenum. Copper absorption from the gut lumen by enterocytes involves both passive and active carrier-mediated systems, which uptake copper across the brush-border, and transport it across the basolateral membrane into the plasma. Most of the copper in foods is found as a component of macromolecules. In- organic mineral salts are present in dietary supplements but otherwise probably do not contribute substantially to dietary copper intake. 64 In the UK only 1–2% of adults report taking supplements containing copper 65 although in the US the figure may be as high as 15%. 17 The sulphate, nitrate, chloride and acetate are easily absorbed, but copper oxide and copper porphyrin are unavailable. 63 Gastric acid can solubilise the carbonate and facilitate the release of copper from macromolecules. 64 Measuring intake of nutrients and their effects: the case of copper 127 Most of the copper in human diets is supplied by vegetable foods, and vege- tarian diets generally provide a higher intake. Plant materials, however, are gen- erally less digestible than animal tissues. A substantial proportion of the copper in whole grains is associated with lectins and glycoproteins. Vegetable tissues fre- quently require more enzymatic attack to digest the copper-binding matrix than do animal proteins, which are generally more easily solubilised, so that percent- age copper absorption may in fact be substantially higher from an animal protein diet than from a plant-protein diet. 66 Even so, the greater copper content of a veg- etarian diet is likely to provide more available copper. 67 Dairy products contain relatively little copper, with cow’s milk being particularly poor. Absorption is estimated at 24% for human milk and 18% for cow’s milk. The quaternary protein structure is thought to exert an effect on the availability of copper in food, as cooked meat has been found to supply more available copper than raw. 64 The efficiency of absorption from food is modified by a variety of luminal factors including copper intake levels, other dietary factors and aspects of the intestinal environment. Dietary components known to modify absorption include protein, amino acids, zinc, manganese, iron, tin, molybdenum, sugars, dietary fibre and ascorbate. 55 Studies of dietary protein and copper retention in young women have found highest retention with a diet high in protein. 68 However, copper bioavailability in high-protein foods may be decreased by heat treatments which promote conden- sation reaction, such as the Maillard Reaction, between sugars and amino acids. 69 The formation of products such as lactulosyl-lysine and lysinoalanine depletes the food of free amino acids. This leaves fewer sites available for the formation of organo-metallic complexes, from which copper is highly bioavailable. 70 The bioavailabilities of copper-lysine and copper-methionine complexes, relative to copper sulphate, have been reported as 120% and 96% respectively. 71 Copper uptake by the intestinal mucosa is strongly influenced by chelation of copper ions by amino acids. Chelation may even be a mandatory requirement for copper absorption. 64 Yet, although dietary amino acids can enhance copper absorption, when present in excess they may result in copper malabsorption, possibly by competing with binding proteins on the enterocyte membrane. The ratio of chelate to metal may determine whether there is a net inhibition or pro- motion of copper uptake. In one human study, methionine supplementation was found to increase copper absorption. 72 Animal studies have provided less straightforward results. One study of rats found that excess dietary methionine decreased indices of copper status. 73 Jejunal copper uptake has been found to be decreased by high levels of dietary proline or histidine, 74 while excessive cystine and cysteine have been shown to exacerbate the effects of dietary copper deficiency. 75 Cysteine is thought to decrease copper bioavailability by reducing Cu (II) to Cu (I). 71 The high bioavailability of copper in human milk, compared to cow’s milk, may be a consequence of the two foods’ protein and amino acid content. Rumi- nant milk has a higher level of low-molecular-weight ligands, which can inhibit copper absorption. In addition, copper is differently-distributed among the milks’ 128 The nutrition handbook for food processors constituents: human milk has a much larger fraction of its copper content bound to whey, as well as to lipids. 64 The antagonistic nature of the copper-zinc relationship has been known for decades. In animals, dietary zinc intake has an inverse relationship with copper absorption. 76 In patients with Wilson’s disease, zinc salts are given orally to lower copper status by limiting absorption. 77 Copper in the gut lumen competes for absorption with zinc, as well as iron and other divalent metal ions. Divalent metals, with their similar electron configurations, can form similar co-ordination complexes. 65 This could reduce absorption by displacing copper from specific transporter molecules on the brush border membrane 78 or by competing for ligands which are necessary for uptake by these receptors. 55 After uptake by enterocytes, intracellular zinc may exert a further antagonistic effect on copper transport. High zinc concentrations are thought to induce the metal-binding protein metallothionein, which has a higher affinity for copper than for zinc. This binding blocks the export of copper, as well as zinc, across the basolateral membrane. 55 Recent studies have elucidated a further aspect of the copper-zinc relationship. 79 Dietary zinc inadequacy was found to be more detrimental to copper status than moderately high zinc intake, suggesting a degree of interdependence. Although at high levels of intake the two metals act antagonistically, adequate zinc levels are beneficial for copper utilisation. Copper status can also be impaired by high intakes of manganese. 80 Iron and tin, in their divalent forms, have also been shown in animals to compete with copper when present in the diet at high levels. 81 Both metals have been known to contaminate food from cooking vessels. Animal studies have suggested that high iron intake affects copper absorption only when copper status is low or mar- ginal. 65 In the context of a copper-normal diet its influence on copper absorption in adults may be minimal. However, babies fed an iron-enriched formula have been found to absorb less copper than infants on the same, but lower-iron, formula. 82 In rhesus monkeys, which are excellent models of human babies, infants fed a commercially-available iron-enriched formula for 5 months had significantly lower copper status than those fed a lower-iron formula. 83 In sheep and other ruminants, interactions between copper and molybdenum have frequently been observed. Chronic molybdenum poisoning in livestock (teart disease) can depress tissue and blood copper levels and produce anaemia and bone deformities, generally symptoms of copper deficiency. In humans, high molybdenum intake has been found to increase urinary copper excretion and result in lowered blood copper. 121 The symptoms of excessive molybdenum intake can generally be improved by increasing copper intake. 122 Molybdenum is also known to influence intestinal copper absorption: the unabsorbable molybdenum complex, thiomolybdate, inhibits intestinal copper uptake and has been used as a treatment for Wilson’s disease. 85 Dietary carbohydrate choice can also influence copper status. The interactions of dietary sugars with copper absorption in humans are not yet well understood, but there is evidence to suggest both systemic and luminal influences upon copper absorption. Glucose polymers are thought to enhance copper uptake Measuring intake of nutrients and their effects: the case of copper 129 by increasing mucosal water uptake. 64 Dietary fibres such as phytate may somewhat decrease copper uptake, but it is likely that other divalent ions are more strongly bound. Dephytinisation, a process frequently used by food proces- sors to improve the bioavailability of metals, can therefore indirectly reduce absorption of dietary copper by increasing the availability of free, competing, divalent ions. 64 In animals, palmitic and stearic acids have been found to reduce the rate of copper uptake from the jejunum. 86 In the literature on humans, there is little data regarding the relationship between dietary lipid intake and copper absorption. One human study of the influence of fatty acids on metal absorption indicated that polyunsaturated fatty acids have no effect on copper uptake. 87 High dietary levels of ascorbate are thought to reduce Cu (II) to Cu (I), thereby lowering its intestinal absorption rate. 88 Conversely, however, utilisation of copper is increased by tissue ascorbate, as it facilitates the release of copper from caerulo- plasmin. 22 High intakes of ascorbate have been found to decrease serum caeru- loplasmin activity and serum copper. 89 A moderately raised intake (605 mg ascorbate/day) has proved sufficient to lower caeruloplasmin activity by 21% without altering intestinal absorption or other markers of copper status. 90 Other organic acids, including citric acid, have also been shown to form soluble com- plexes with copper. It is probably for this reason that fruit intake has a positive effect on copper status. 91 The efficiency with which any dietary nutrient is absorbed and utilised in the body is described as bioavailability. It is an essen- tial consideration in the nutritional evaluation of foods and diets. 12 In studies of the bioavailability of some minerals, the degree of utilisation may be inferred by measuring some functional endpoint such as the level of synthe- sis, or activity, of certain biomolecules. Iron bioavailability, for instance, can be determined by measuring the incorporation of a stable isotope into haemoglobin. For copper, however, no single index of utilisation has yet been identified. As a result, estimates of bioavailability have previously focused on measuring intesti- nal absorption, or bodily retention, rather than utilisation. 92 Nonetheless, copper utilisation is influenced by a number of endogenous factors not directly related to luminal absorption rates. By exerting nonluminal effects upon copper utilisa- tion, such factors may result in impaired copper status. The copper-depleting effect of excess dietary histidine in rats is associated with increased urinary excretion of chelated copper. 93 The high level of low- molecular-weight chelates in cow’s milk may help to explain the copper defi- ciency sometimes observed in infants fed on unmodified cow’s milk. This may be particularly relevant during periods of anabolic activity, such as recovering from malnutrition. An association has also been observed between very high intake of fructose or sucrose and a worsening of the effects of copper deficiency in rats, 94,95 but not in pigs. 96 In humans, similar experiments 97 have produced changes including cardiac arrhythmia and reduction of erythrocyte SOD activity with apparently increased copper balance, suggesting that high fructose intake acts systemically to raise body copper requirements. Experimental evidence implicating high fat 130 The nutrition handbook for food processors intake as a further aggravating factor 98 suggests that the fructose-copper interac- tion may be associated with altered energy metabolism. 64 5.11 Copper distribution in the body Copper distribution around the body appears to operate in two phases. 60 In the first phase, copper ions are exported from enterocytes into the circulation. This is controlled by specific copper transporting proteins, including ATP7A, a P-type ATPase localised to the trans-Golgi network. It is also known as the Menkes protein MNK, because hereditary deficiency results in Menkes disease. 99 Copper ions secreted from the intestinal mucosa are immediately bound to the high-affinity plasma proteins albumin and transcuprein. 60 In physiological condi- tions, copper is almost always protein-bound, resulting in extremely low plasma concentrations of free ionic copper, perhaps as low as 10 -18 Molar. Protein-bound copper is transported to the liver and kidney. Of the copper taken up by liver parenchymal cells, approximately 80% is excreted in the bile. 100 Intestinal excre- tion provides the major mechanism for body copper homeostasis, urinary copper excretion being normally less than 0.1 mg/d. Several pathways involving sequen- tial protein-to-protein transfers are believed to be involved in copper transport across the hepatocyte. 101 Cytoplasmic carrier proteins deliver copper to sites of synthesis of cytoplasmic cuproenzymes such as superoxide dismutase. Carrier proteins also supply copper to specific organelle-bound transporter proteins which control its incorporation into mitochondrial proteins, or entry into the hepatocyte secretory pathway. 60 The trans-Golgi network protein ATP7B is involved both in bile formation and in caeruloplasmin secretion. 99 Congenital deficiency of this enzyme results in Wilson’s disease. The second phase of body copper transport involves its efflux from liver and kidney and delivery to other tissues. It is secreted into the plasma, from liver and kidney cells, bound mainly to caeruloplasmin. 60 From studies using radioisotopes to trace body copper transport, caeruloplasmin appears to be the main copper carrier around the body. Most tissues have been shown to have specific surface receptors for caeruloplasmin-copper, and to take it up from solution, 102 either by endocytosis of the complex or by transfer of the copper to an intracellular receptor. There remains some uncertainty, however, concerning the importance of caeruloplasmin’s role in body copper distribution. The protein is not thought to be crucial to copper transport because the genetic defect acaeruloplasminaemia does not severely disrupt copper metabolism. It appears that there is redundancy in the system, with copper also available to tissues from non-caeruloplasmin sources including proteins and other ligands. Current thinking is that tissues absorb copper preferentially from caeruloplasmin, but can utilise other sources if caeruloplasmin is not available. 60 Hephaestin is a recently discovered membrane-bound glycoprotein. A homo- logue of caeruloplasmin, it appears to play a role in iron metabolism and has been most highly localised to the small intestinal villi, the site of iron absorption. 103 Measuring intake of nutrients and their effects: the case of copper 131 5.12 Assessment of copper absorption Early research into human copper metabolism involved studies of copper intake and excretion, copper balance and tissue concentrations, which permitted the esti- mation of bodily requirements and dietary recommendations. 104 Studies on labo- ratory animals, and the use of in vitro techniques such as intestinal perfusion and the creation of sacs from everted duodenal segments, have contributed much to our understanding of intestinal mechanisms. The use of balance studies to examine copper metabolism poses certain difficulties. Firstly, the regulation of copper absorption according to dietary intake is a process which may require a period of adaptation. For absorption to reflect bodily requirement accurately, therefore, a balance study must be of considerable duration. 92 Secondly, the estimation of losses is difficult owing to a current scarcity of data concerning copper levels in sweat, integument, hair, nails, menstrual blood and semen. Thirdly, balance studies of children must account for the changing copper require- ments associated with growth, about which little information is available. 105 The introduction of isotopic tracers as an investigative tool has permitted detailed examination of absorption mechanisms, dose effects and interactions with other minerals and food components. Findings in mammals of a saturable, carrier-mediated transport mechanism are compatible with the dose-related reduc- tion in absorption which has been demonstrated in humans. The absorption of stable and radioactive isotopes of copper may be determined after oral adminis- tration by monitoring either their disappearance from the gut lumen or their incor- poration into biomolecules. 106 Copper has seven radioisotopes, of which only 64 Cu and 67 Cu have half-lives long enough to be useful in metabolic research – 12.8 h and 58.5 h respectively. These relatively short half-lives limit the use of radioiso- topes to short-term studies. Longer-term studies require the use of stable isotopes which have several additional advantages: because they emit no radiation, they are safe to use in high-risk population groups; and because there is no decay, samples can be stored without loss of signal. Copper has two stable isotopes, 63 Cu and 65 Cu, which both have high natural abundances – 69.2% and 30.8% respectively. To act as a tracer, an isotope must be ‘enriched’ to a higher proportion than in nature. The pro- duction of enough 63 Cu or 65 Cu to detect above background levels is costly. The use of such large doses raises further problems. The intravenous administration of non-physiological quantities of the mineral may alter normal metabolism 107 while the labelling of food with 65 Cu has been found to change its copper content substantially. 108 In studies of trace minerals, the simultaneous use of multiple stable isotopes offers a means to study the effects of different compounds and different routes of administration. Such studies are necessarily impossible for copper, because with only two stable isotopes, only one can be enriched at a time. Biological samples obtained in stable isotope studies are analysed by deter- mining isotopic ratios. Available methods are generally slow and expensive, and require access to sophisticated analytical equipment. Neutron Activation 132 The nutrition handbook for food processors Analysis, Electron Ionisation Mass-Spectrometry and Gas-Chromatography Mass-Spectrometry all offer relatively poor precision, while Thermal Ionisation Mass-Spectrometry is laborious and slow. Inductively Coupled Plasma Mass- Spectrometry, used since the 1980s for trace-element quantification, offers acceptable precision with faster analysis and a lower limit of detection than the other methods. 109 Most radioisotopes can be measured by whole-body counting of gamma-emissions, but this method of detection is not readily applicable to copper, owing to the radioisotopes’ short half-life. It has, however, been applied in studies of abnormal copper absorption and retention. 110 Faecal monitoring, of stable or radioisotopes, is currently the most widely used method for assessing copper absorption. A stool marker may be given simulta- neously to test for completeness of faecal collection, and may consist of indi- gestible beads or a non-absorbable chemical marker. In this method, the relatively rapid re-excretion of absorbed copper necessitates special consideration. Even before the non-absorbed fraction of an oral dose has left the body – a process which has been found to take five to seven days – re-excretion of the absorbed isotope will have begun. To correct for this, the rate of endogenous excretion must be determined. Owing to the large inter-individual variation, 110 it should be measured in each individual. Faecal monitoring of a radioisotope for longer than five days would require more than the maximum safe dose 106 so endogenous excretion must be determined on a separate occasion using an intravenous dose. One application of tracer data obtained from isotope studies is the develop- ment of compartmental models of metabolism. In this technique, modelling soft- ware is used to compile extensive data on copper distribution and transport into a model simulating whole-body copper metabolism. This provides a powerful tool to describe and predict copper kinetics and to determine dietary require- ments. 111 Kinetic modelling provides a means to correlate experimental data from previous studies. Existing information including tissue concentrations, fractional transfer and turnover rates can be assembled into a system in which known com- ponents are viewed in perspective. This can have the effect of highlighting areas requiring further research. It can also be used to improve experimental design by simulating in advance the system of interest. 5.13 Current research and future trends Research into copper metabolism has benefited from recent advances in several areas, with development of novel techniques and refinement of existing methods for the measurement of copper absorption, utilisation and excretion; and ongoing investigations into the biological roles of copper. A recent development in faecal monitoring techniques has been the validation of a novel method to distinguish the non-absorbed portion of an oral label from the absorbed but re-excreted portion. 112 While, previously, this was achieved by a separate test of endogenous excretion rate, it is now possible to measure true absorption by use of the rare earth metal holmium as a faecal marker. Because its excretion pattern parallels Measuring intake of nutrients and their effects: the case of copper 133 that of copper, it can safely be assumed that all label recovered after complete holmium clearance, is re-excreted copper. While other rare earth metals have been used to check for completeness of faecal collection, their excretion pattern differs from that of copper, precluding their use for estimating true absorption. Owing to the limitations of faecal collection, a plasma indicator of absorption may be pre- ferable. This approach requires that newly-absorbed, albumin- and transcuprein- bound copper be distinguishable from the caeruloplasmin-copper pool. Whereas the movement of injected isotopes between the two compartments is readily trace- able, orally-administered isotopes are more slowly transported into plasma, resulting in a problematic temporal overlap of the two copper pools. This issue has recently been addressed in a novel method of separating tracer-bound albumin by dialysis. 113 Recent research using both stable and radioisotopes has benefited from the development of detectors with ever-lower detection limits and the ability to dis- tinguish different isotopes. An increasing range of software applications for the mathematical modelling of biological systems has also contributed to ongoing developments. One such application is SAAMII, produced in the University of Washington, Seattle.Arecent mass spectrometry technique currently being applied to human nutrition studies is Inductively-Coupled Plasma Mass-Spectrometry. With a reported lower limit of detection below 1.4 ng/ml human plasma, 114 it has recently been used to evaluate apparent copper absorption from vegetarian and non-vegetarian diets. 67 Accurate assessment of functional copper status has long been hindered by the current lack of a plasma or tissue parameter suitable for use as an index of copper utilisation. Ongoing investigations attempt to identify such a marker. Although numerous copper-containing biomolecules have been observed to change in response to clinical copper deficiency, none has yet been verified as a valid index of marginal copper status. PAM, DbM and tyrosine mono-oxygenase have all been indicated in animal studies as potential markers of marginal copper status. 115,116 PAM activity in Menkes patients is reduced, but is modifiable in vitro by addition of copper, thereby obtaining a copper stimulation index. This technique could provide an indicator of copper status, but requires validation in human trials. Animal studies have suggested tissue activities of cytochrome-c oxidase and lysyl oxidase as early indicators of copper deficiency. 53,117 Experimental copper depletion in humans has produced decreased cytochrome-c oxidase activity in leucocytes and in platelets, but the latter measure has sometimes failed to respond to copper repletion. 56 In human skin, lysyl oxidase activity has been seen to decline with copper depletion and to respond to copper repletion. 118 Too few data are currently available to determine whether these enzymes may be feasible indices of mar- ginal copper status. In addition, the invasive nature of biopsy makes tissue enzymes assays undesirable for general use. Measurements of blood copper concentration and cuproenzyme activities have not so far proved to be a sensitive method for the evaluation of nutritional status at the tissue, organ and systemic levels. Tissue sampling is generally not a 134 The nutrition handbook for food processors feasible option for use in the general population. One suggested alternative to biochemical testing is the measurement of some other aspect of biological func- tion which is known to be dependent on copper sufficiency. If the adequacy of response to a stressor is regulated by copper status, then response will be adverse or deleterious when status is suboptimal. 119 Potential parameters for functional testing may, in theory, include any aspect of physiological or psychological func- tion which can be shown to be altered by copper depletion and supplementation. One candidate is blood-pressure response to isometric work. In young women undertaking a standardised hand-grip exercise, the blood pres- sure response was exaggerated when dietary copper was restricted to 0.65 mg/d. 34 This was observed in the absence of significant changes in copper balance or plasma copper, but with reduced caeruloplasmin activity. Good correlations were found, for individual subjects, between blood pressure response and caeru- loplasmin concentration, demonstrating a relationship between the biochemical and physiological indices of copper status. The application of performance- related indices in parallel with biochemical measurements allows potential novel indicators of copper status to be evaluated. Recent research into the stimulatory effect of copper deficiency upon hepatic lipid synthesis has examined the mechanisms behind the observed increase in transcription of lipogenic gene expression. 120 Findings suggest that copper defi- ciency stimulates the expression of the fatty acid synthase gene by increasing the nuclear localization of a mature transcription factor, sterol regulatory element binding protein-1. There is clearly a need to identify more closely the role of copper in biologi- cal systems, both in health and in disease states. According to the WHO, there is a great need for standardised sampling and analytical procedures for the deter- mination of dietary copper and copper in drinking water. There appears, also, to be a case for revision of the existing guidelines for copper in drinking water. Whereas the US EPA has used acute toxicity data to derive its guideline, the WHO has used data on total copper intake and chronic toxicity. A greater degree of con- sensus on the criteria used would be instrumental in the establishment of sounder, more defensible guidelines. In Western societies an emerging issue is the identification of the require- ments for optimal nutrition. The importance of developing such markers can hardly be overemphasised. With these tools, appropriate studies can be used to establish recommendations for optimal copper intake of individuals and of populations. 5.14 Sources of further information and advice World Health Organization: http://www.who.int/home-page/ International Programme on Chemical Safety: http://www.inchem.org European Commission Scientific Measuring intake of nutrients and their effects: the case of copper 135 Committee on Food: http://www.europa.eu.int/comm/food/index_en.html UK Food Standards Agency: http://www.food.gov.uk/ The Nutrition Society: http://www.nutsoc.org.uk/ US National Institute of Medicine, Food and Nutrition Board: http://www.nationalacademies.org/sitemap/ Ministry of Agriculture, Fisheries and Food: http://archive.food.gov.uk/dept_health/pdf/evmpdf/erm9919.pdf 5.15 References 1 hart e b, steenbock h, waddell j and elvenhjem c a (1928), ‘Iron Nutrition. VII. 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