5 Measuring intake of nutrients and their effects: the case of copper L B. McAnena and J M. OConnor, 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; nd 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 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 recommendations, 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 associated 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
118 The nutrition handbook for food processo 5.2 The nutritional role of copper Copper was identified as an essential trace element, first for animals and sub sequently for humans- when anaemia was successfully treated by supplementin 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 Table 5.1 Human copper-containing proteins, and their functions Protein Function Cytochrome-c oxidase Cellular energy production Ferroxidase I(Caeruloplasmin ron oxidation and transport; free radical avenging; amine and phenol oxidation acute-phase immune res Ferroxidase ll ron oxidatio Hephaestin Iron metabolism Copper/zinc superoxide dismutase Antioxidant defence Extracellular superoxide dismutase Antioxidant defence Monoamine oxidase Brain chemistr Dopamine B-hydroxylase Brain chemistry Diamine oxidase Limitation of cell growth, histamine deactivation Lysyl oxidase Connective tissue formation Peptidylglycine a-amid Peptide hormone activation Prion protein PrP Antioxidant defence and/or copper sequestration and transport Tyrosine Melanin synthesis Albumin Metal binding in plasma and interstitial fluids Chaperone proteins intracellular c tar proteins Chromatin scaffold protein Structural integrity of nuclear material Clotting factors v and vIll Thrombogenesis Metallothionein Metal sequestration Copper binding in plas
5.2 The nutritional role of copper Copper was identified as an essential trace element, first for animals1 and subsequently for humans2 when anaemia was successfully treated by supplementing the diet with a source of copper. Since then the full significance of its role in biological 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. Functional 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 proteins 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 -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
Measuring intake of nutrients and their effects: the case of copper 119 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. 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 Il are plasma glycoproteins Ferroxidase I, also known as caeruloplasmin, oxidises Fe (m) to Fe (im) without formation of hydrogen per- oxide(H2O2)or oxygen radicals. It is primarily this role which gives rise to caeru- loplasmin's well-known antioxidant function. It also scavenges H2O2, superoxide and hydroxyl radicals, and inhibits lipid peroxidation and DNA degradation stimulated by free iron and copper ions. 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 enzymes catalytic action. 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 SoDI present in most cells; and the extracellular SOD2, found in the plasma and also in certain cell types in the lung, thyroid and uterus. 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 nd 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. 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
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 peroxide (H2O2) or oxygen radicals. It is primarily this role which gives rise to caeruloplasmin’s well-known antioxidant function. It also scavenges H2O2, 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 participate 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 wellstudied 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 dismutation 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) inactivates, by deamination, substrates such as serotonin and catecholamines including 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 monooxygenase 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
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 Food and Nutrition Board, 2001 WHO AROI 1.2 to 2 or 3 WHO International Programme of Chemical Safety, 1998 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. 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, 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 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 ufficiently sensitive, specific and consistent to be used alone. A Recommended Daily Allowance(RDA)can be calculated by extrapolatin the EAr to account for inter-individual variation in requirements. The US RDA, te the UK Reference Nutrient Intake (RND) is intended to provide enough copper for about 97% of adults. The World Health Organization has loosely defined an Acceptable Range of Oral Intake(AROD). 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 ecause 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-
leading to melanins occur spontaneously in vitro. Regulation of pigment formation 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 mechanisms, 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 recommendations, even of Estimated Average Requirements (EAR), is difficult owing to a lack of adequate data. In the UK, the Department of Health considers the available 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 biochemical 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
Measuring intake of nutrients and their effects: the case of copper 121 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 dequate to meet the body's requirements Published estimates of copper intake vary around 1-2mg/d, with few diets containing more than 2 mg/d. 3.1415, 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. Copper deficiency during this period has been shown to impair growth rate and to be associated with increased incidence of respiratory infection. 9 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 Although neonates appear not to absorb copper well, particularly from highly refined carbohydrate-based diets or cows milk, full-term infants have well leveloped copper stores which can be mobilised during the first six months rapid growth, to supplement dietary intake. Full-term infants are therefore independent of dietary intake for the first weeks of life. 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. 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 intestinal copper losses leading to deficiency. Such conditions include coeliac disease26, cystic fibrosis, shortened intestine following surgery, 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
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. Nonetheless, 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 children, 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. Malnourished children are reported to be at particular risk of copper deficiency. A diet consisting exclusively or predominantly of cow’s milk, with its poor bioavailability of copper, increases the likelihood of copper malabsorption. During nutritional 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 rate18 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 metallothioneinbound copper accumulates in the foetal hepatocyte nuclei over the last trimester.11 Although neonates appear not to absorb copper well, particularly from highlyrefined carbohydrate-based diets or cow’s milk20, full-term infants have welldeveloped 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 interactions 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 disease26, cystic fibrosis27, shortened intestine following surgery28, 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