Copper

I.  Physiological Role of Cu (also see Nutr. Revs. 43:117. 1985)

1. Copper dependent metalloproteins
   1. Cytochrome C oxidase - Part of the electron transfer chain
   2. Dopamine b-hydroxylase catalyzes conversion of dopamine to norepinephrine.  Dopamine
      b-monoxygenase is a cuproenzyme that catalyzes formation of norepinephrine from dopamine. (J. Nutr. 120:88 1990)
   3. Monoamine oxidase
      1. Molecular weight 195,000 daltons, contains 4 atoms Cu/molecule
      2. Catalyzes oxidative deamination of a variety of monoamines to the corresponding aldehydes and is involved in maintaining structural integrity of both vascular and bone tissue
   4. Ceruloplasmin, a Cu-transport protein, also has other functions
      1. A ferroxidase which catalyzes oxidation of Fe (Fe⁺²® Fe⁺³) for binding to plasma transferrin; required for Fe absorption and for hemoglobin formation
      2. Participates in the utilization of Cu ²⁺ for the biosynthesis of cytochrome C oxidase and other Cu proteins
      3. May help regulate plasma biogenic amines through its oxidative action on the epinephrine and serotonin series
   5. Urate oxidase - oxidation of uric acid to allantoin
      Uric acid + 0₂ + 3H₂0 ® Allatoin + H₂0₂ + HCO₃^-
   6. Lysyl oxidase - an amine oxidase necessary for formation of crosslinking compounds in collagen and elastin. It is involved in maintaining structural integrity of both vascular and bone tissue
   7. Tryosinase - essential in the pigmentation process since it catalyzes the first two steps in the synthesis of melanin from tryosine
   8. Superoxide dismutase - protection from oxidants. (Hepatocuprein, erythrocuprein, cerebrocuprein) catalyzes the reaction
      20₂^- + 2H⁺  ® 0₂ + H₂0₂
      1. A link in the chain which protects cells against superoxide ions, an unavoidable byproduct of oxygen reduction
      2. H₂0₂ is also cytotoxic and is controlled by other links in the chain (glutathione peroxidase,  catalase)
      3. Strict anaerobes do not have SOD. Its absence may explain their vulnerability to oxygen
2. Cu is involved in prostaglandin synthesis (Biochem. Biophys. Acta 306:74. 1973)
3. Component of adenine nucleotide binding sites (Aust. J. Exp. Biol. Med. Sci. 54:593. 1976).  ATP binding is depressed by Cu-complexing agents and increased when mitochondrial membrane Cu content is increased

II.  Tissue Distribution of Cu

1. Plasma (or serum) Cu (mg/dl) Man, 120; horse, 90; cow, 80; sheep, 120; pig, 160; rat 200
   1. Ionic Cu is so toxic it cannot exist free in the body in substantial amounts so it must be delivered to Cu enzymes and other sites of action via a series of binding molecules.
      1. Metallothionein in intestinal tissue may regulate the quantity of Cu that passes into the blood subsequent to acute ingestion of Cu
      2. Cu-albumin and Cu-histidine are transient absorption forms on their way to the liver for storage and/or biosynthesis of ceruloplasmin
      3. Ceruloplasmin, the principal Cu protein in plasma is an aglobulin complex containing 8 atoms of Cu. Its ability to serve as a transport vehicle and release Cu at specific cellular sites has been related to its catalytic activity as an oxidase in which Cu cycles from Cu²⁺ to Cu¹⁺ and back again. Ascorbate may be a cofactor or catalyst in the cross-membrane exchange of Cu from ceruloplasmin to the tissues (J. Nutr. 119:779-784. 1989)
   2. Disease conditions (acute arthritis, infected wounds, meningitis) may increase plasma Cu (ceruloplasmin)
   3. Elevated plasma Cu has also been noted in animals with hypocalcemia, hypomagnesemia and pregnancy toxemia (because of degenerative changes in the liver?)
2. Erythrocytes
   1. Cu is found in RBC in a nearly colorless protein complex called erythrocuprein
      1. Molecular weight of about 35,000
      2. Contains about 0.34% Cu
   2. Erythrocuprein has superoxide dismutase activity
3. Liver
   1. Liver is the major storage organ for Cu
   2. Much of the stored Cu is attached to metallothionein
   3. About half of the total body Cu of the newborn is in the liver
      1. As a Cu reserve for newborn when they are receiving milk
      2. Is capacity of the fetus to excrete Cu limited?
      3. Is the fetus unable to synthesize ceruloplasmin?
4. Cu averages near 10 mg/dl in ovine (J. Anim. Sci. 59:41 6. 1984) or bovine milk

III.  Absorption and Excretion of Cu

1. Cu is required for many biological functions but its chemical properties render it extremely damaging to biological systems
   1. Animals possess specific mechanisms to channel proper amounts of Cu to correct physiological locations for its specific functions
   2. The delicate balance between essentiality and toxicity is influenced by ligands with which Cu and other metals associate. (Ligands are defined as neutral or negatively charged molecules that surround a central metal via specific binding forces)
2. Absorption occurs in the duodenum and jejunum
3. Excretion is primarilyy via feces

IV.  Metallothionein (from Physiol. Revs. 65:238. 1985)

1. Isolated and characterized by Kogi and Vallee (1960. J. Biol. Chem. 235:3460) who were searching for a reason for accumulation of nonnutrient metals (mercury, cadmium) in the tissues
2. Characteristics of metallothionein
   1. A single polypeptide chain of 61 amino acids
   2. 25-30% of the amino acid residues are cysteine, and in the native protein, there are no disulfide bonds.
   3. No aromatic amino acids occur in the peptide
   4. The protein exhibits a significant degree of polymorphism
   5. Sequence homology is excellent, indicating a highly conserved primary structure.
3. It has a metal-binding capacity of 5-7g atoms/mol
   1. Cysteine residues provide the necessary metal-binding ligands
   2. Each metal ion is arranged in a tetrahedral coordination complex with four thiolate ligands.
   3. The seven metals (can be a mixture) bound to metallothionein are arranged in two clusters, one with 4 atoms (cluster A) and another with 3 atoms (cluster B)
   4. Metal binding occurs first in cluster A, followed cooperatively by cluster B
   5. The order of release of metals may be the reverse of binding
   6. Metallothionein binds both Zn and Cu under physiological conditions, so it probably is involved in metabolism of both
   7. When Cu is bound, the coordination arrangement is probably different and may involve more atoms per mole. This could account for the markedly different metabolic properties of Cu-rich metallothionein

V.  Homeostatic Control of Cu

1. Regulation of Cu absorption is the primary control
   1. Metallothionein in the cytosol of intestinal cells may regulate the quantity of Cu that passes into the blood stream
   2. Ruminants are particularly susceptible to Cu poisoning because control of Cu absorption is limited
      1. In sheep, unlike other species, Cu is not associated with metallothionein in intestinal mucosal cytosol
      2. Sheep's capacity to synthesize metallothionein in response to increased dietary Cu is limited
      3. This may contribute to the sheep's susceptibility to Cu poisoning
      4. Sheep store excess Cu in the liver. Sudden breakdown of this storage releases Cu from the liver into the blood causing the "hemolytic crisis"
2. Excretion of Cu
   1. Excretion of Cu in the bile may be a principal homeostatic control mechanism for Cu in nonruminants
      1. In nonruminants, Cu is excreted in bile via sequestration of protein-bound Cu (metallothionein) by the lysosomes
      2. Cu excreted into the small intestine in bile is poorly reabsorbed
      3. Urinary Cu excretion is low in nonruminants
3. Storage of Cu in the liver (hepatic metallothionein?)
   1. Pigs have an unusually high storage capacity for Cu in the liver, lungs and kidneys. A high Cu concentration in these organs does not seem to have a deletrious effect
   2. Cu toxicity in pigs is low compared to other species
   3. Increased Cu intake increases Cu turnover in various organs of the pig
   4. Sheep can store Cu in the liver up to a point

VI.  Interactions with Other Elements (See Suttle, N.F. 1991. The interactions between copper, molybdenum, and sulfur in ruminant nutrition. Ann. Rev. Nutr. 11:121-140)

1. Sulfur and molybdenum in ruminants
   1. S and Mo within normal dietary range (0.1 - 0.4% S and 0.4 - 4.5 ppm Mo, dry basis, can affect Cu availability
      1. S exerts a predominant and independent effect
      2. Mo has a lesser and S-dependent effect
      3. Mo depletes liver Cu severely only when adequate S is present
   2. Both S and Mo must be elevated for sheep to develop dystrophic wool (a sign of Cu deficiency)
   3. Mo and S together elevate plasma total Cu without affecting ceruloplasmin
   4. Increase is due to TCA insoluble Cu.
   5. Several mechanisms have been proposed to explain the 3-way interaction
      1. Blocking of Cu transport across membranes
      2. Competition for a common carrier system
      3. Lower Cu absorption due to insoluble Cu sulfides or Cu-Mo complexes
      4. These mechanisms are all consistent with reduced liver Cu but why is plasma Cu elevated?
2. Effect of other dietary components
   1. Selenium: different effects have been reported in ruminants and nonruminants
      1. Small oral doses of Se improved growth and tissue Cu in lambs
      2. Rats fed diets low in Se appear to have a lowered threshold of Cu toxicity
   2. Manganese: Supplemental Mn may also improve Cu absorption in lambs
   3. Iron: Ceruloplasmin is a molecular link between Cu and Fe metabolism
      1. Cu, as ceruloplasmin, is necessary for Fe utilization
      2. Plasma Fe of Cu deficient lambs is often low
      3. Excess dietary Fe (800 ppm) can reduce plasma ceruloplasmin in calves to levels indicative of severe Cu deficiency
   4. Cobalt: Supplemental Co for lambs with Co deficiency combined with Mo/S induced Cu deficiency, reduced gains and lowered liver Cu.  In rat, cobalt increased urinary Cu excretion 4-fold (J. Nutr. 119:1259-1268. 1989)
   5. Zinc: Zn at 10x dietary requirement reduces Cu toxicity
      1. Reduces total Cu content of liver
      2. Increases Cu content of sheep liver metallothionein
   6. Tin: Rats fed high Sn diets accumulated less Cu in plasma, kidney & liver (J. Nutr. 115:615. 1985)
   7. Magnesium. Fertility of cows was improved when they were supplemented with both Mg and Cu but not with Cu or Mg alone (J. Dairy Sci. 70:167.1987)
      

      Control	Cu	Mg	Cu+	Mg
      First conception rate (%) Conception by 150d postpartum (%) Services per conception (no.)	33 59 1.9	27 62 1.9	38 63 2.0	57 84 1.6

   8. Cadmium: Cu absorption is decreased when supplemental Cd is fed.  Decreased Cu absorption is associated with Cu incorporation into metallothionein induced by Cd
   9. Protein: Solubility of Cu in rumen and abomasal contents decreases proportionally with increasing dietary protein
   10. Ascorbic acid (J. Nutr. 117:2109-2115. 1987)
       1. Papers are cited which showed apparent antagonism of ascorbic acid toward Cu metabolism
       2. In humans, moderate intakes of ascorbic acid reduced ceruloplasmin oxidase activity specifically
       3. However, ascorbic acid did not depress Cu absorption or overall body Cu status
   11. Ascorbate enhanced uptake of Cu by cells from ceruloplasmin. (J. NUtr. 119:779-784, 1989).  Ascorbate may play a role in ceruloplasmins delivery of Cu to extrahepatic tissues
   12. Dietary fructose increases severity of Cu deficiency. Ingestion of fructose as compared to starch increases Cu requirement. (J. Nutr. 119:453-457. 1989)

VII.  Cu Requirements

1. If the Natl. Acad. Sci. recommendation of 2-3 mg Cu/d for adult human is correct, intakes of both men and women reported by Patterson et al. (Am. J. Clin. Nutr. 40:1297-1403. 1984) are low. They reported 1.4 mg/d for males and 1.1 mg/d for females.
2. Estimation of requirements
   1. Subjects are fed different amounts of Cu and some function of absorption or retention which when regressed on intake gives a straight line is determined.
      1. Maintenance = intake at 0 retention
      2. Endogenous loss - retention at 0 intake
   2. Suttle determined Cu requirements of cattle by an IV repletion technique based on the assumption that total net Cu requirement approximates the daily rate at which the injected dose of Cu is used by calves on an extremely Cu deficient diet
3. Measurement of Cu status
   1. Liver Cu is a good criterion of Cu status.
   2. Blood plasma is more easily obtained and can be used to indicate a deficiency but it does not closely reflect higher Cu stores in liver
   3. Serum ceruloplasmin is often used
4. Recommended allowances of Cu
   1. Human...............................2 mg/d
   2. Swine & poultry rations.....4 to 5 ppm
   3. Ruminant rations...............8 to 20 ppm
      1. Adequate when dietary conditions are optimal for utilization of Cu
      2. Requirement for ruminants may be increased by dietary Mo differences of only 1 ppm

VIII.  Dietary Sources of Cu

1. Most feeds. Plant materials contain Cu which has an affinity for-plant lipids
   1. Cu content of oil seed meals is relatively high (50-100 ppm).
   2. There is evidence that water-soluble complexes of Cu in herbage are used more efficiently by the rat than are many inorganic sources
   3. Cu proteinate may be used more efficiently than inorganic sources by cattle when excess Mo is a problem
   4. Cu availability to ruminants is closely related to forage quality
2. Inorganic Cu salts (in order of availability)- Cu chloride, sulfate, nitrate, carbonate, oxide
3. With high dietary Mo, Cu may be more available from the proteinate than from the sulfate

IX.  Cu Deficiency

1. Acute diarrhea can deplete Cu in infants (Nutr. Rev. 48:19. 1990)
2. Human diseases associated with hypocupremia
   1. Menkes syndrome
      1. Lethal X-linked inherited neurodegenerative disease
      2. Results from a defect in the low molecular weight ligand that transports Cu into cells
      3. Leads to accumulation of excessive Cu in (or on) the cells
      4. Overall Cu deficiency is imposed on this situation
   2. Wilson's disease
      1. Failure of use of Cu for ceruloplasmin biosynthesis
      2. Cu accumulates in the liver and brain but is lacking in other tissues
3. Cu status of human subjects decreased by Zn supplements (Fischer et al., 1984. Am. J. Clin. Nutr. 40:743-746)
   1. Plasma Cu levels or ceruloplasmin were not affected
      1. For ferroxidase (ceruloplasmin) activity to be lost, two of the six Cu atoms associated with ceruloplasmin must be lost
      2. Since most of the plasma Cu is associated with ceruloplasmin, and since plasma Cu did not change, sufficient Cu was available to maintain ferroxidase activity
   2. Plasma Zn was increased
   3. Cu, Zn-superoxide dismutase decreased after 6 wk
      1. Cu is added to the apoenzyme superoxide dismutase at the time of erythropoiesis only
      2. If Cu is not lost from SOD during the lifespan of the erythrocyte, then it is likely Cu status for Zn supplemented individuals decreased well before the 6 wk without being apparent, due to the-long half-life of erythrocyte (only newly formed RBC would reflect lower Cu status)
   4. These results support the hypothesis that functional nutritional indices, such as SOD activity in the case of Cu status, are more sensitive than static indicators, such as plasma or tissue Cu levels
      • See J. Nutr. 118:859, 1988. In studies with rats, a 10-fold reduction in hepatic Cu only resulted in a 2-fold reduction in SOD activity. In this regard, SOD appears to be given high priority with respect to the utilization of cellular Cu
      • See J. Nutr. 120:88,1990. Cu deficient rats has smaller norephinephrine pool possibly due to limiting dopamine-b-monooxygenase activity
      • See J. Nutr. 119:1259-1268, 1989. Co increased urinary excretion of Cu 4-fold without an accompanying loss of Zn such as accompanies use of pennicillamine. Zn retention occurred with Co treatment, most likely due to cobalt mediated induction of metallothionein in the liver. Use of small amounts of Co may have clinical potential in elm·nation of Cu in Cu-overload disorders.
4. Effects of Cu and/or Fe deficiency on Fe utilization by rats (Cohen et al., 198~. J. Nutr. 115:633-649) Data from 49th day
   

   Dietary		Status	Plasma		Liver		Ferroxidase (ceruloplasmin)	Ascorbate	Hemoglobin	Liver Xanthine Oxidase
   Fe	Cu	Fe/Cu	Cu	Fe	Cu	Fe
   			mg/g wet		IU/mol		Mg/dl
   120 25 10 120 25 10	10 10 10 0 0 0	A/A M/A L/A A/D M/D L/D	1.20 1.05 0.95 0.06 0.05 0.03	4.36 2.67 0.91 2.37 1.48 0.45	3.82 3.81 4.14 1.11 1.01 0.62	230.1 101.9 31.9 279.9 184.6 90.8	8.32 8.26 8.37 0.02 0.08 0.04	0.98 0.96 1.25 0.62 0.59 0.97	15.1 15.1 10.3 14.9 12.6 8.3	5.3 5.5 5.8 3.7 3.4 3.7

   1. At each dietary Fe level, plasma Fe was lower, but liver Fe was higher when Cu was deficient.
   2. Ferroxidase activity of Cu deficient groups was < 10% that of Cu-sufficient groups.
   3. When low or marginal levels of Fe were fed, hemoglobin levels in Cu deficient groups were lower than in Cu sufficient groups. When Fe was adequate, hemoglobin was similar in Cu deficient and Cu sufficient groups
   4. Cu deficiency tended to result in lower concentrations of ascorbate and activities of xanthine dehydrogenase
   5. These results show that Cu deficiency may impair liver Fe mobilization in the growing rat if dietary Fe is low
   6. Possible mechanisms include decreasd ferroxidase activity and/or decreased iron reduction by ascorbate or xanthine dehydrogenase
5. Cu deficiency symptoms
   1. Growth retardation
   2. Impaired feed conversion
   3. Diarrhea
   4. Rough hair coat; loss of crimp in wool due to loss of disulfide bond formation
   5. Faulty connective tissue due to lack of lysyl oxidase
      1. Leg abnormalities
      2. Thin, fragile bones that break easily
      3. Massive internal hemorrhages resulting from spontaneous rupture of a major blood vessel (falling disease)
   6. Anemia
   7. Reduced serum Cu and ceruloplasmin
   8. Reduced Cu and cytochrome oxidase in brain and liver tissue
      Increased liver Fe deposition
      Lack of pigmentation (decreased tyrosinase activity)
   9. Nervous disorders, neonatal ataxia in lambs - lack of myelination in the spinal cord

X.  Cu Toxicity

1. Nonruminants can tolerate more Cu than ruminants and effects of Cu toxicosis are less dramatic
   1. Maximum tolerable levels (NRC, 1980)
      1. Swine............250 ppm (Cu at this level can be growth stimulant)
      2. Horses..........800 ppm
      3. Chickens......300 ppm
      4. Turkey..........300 ppm
      5. Rabbit...........200 ppm
      6. Rats..............1,000 ppm
      7. Human..........10 mg/d
   2. Toxicity signs in nonruminants: Growth inhibition, anemia, muscular dystrophy, impaired reproduction, decreased longevity
   3. The range between inadequate and excessive Cu for ruminants can be relatively narrow. Maximum tolerable levels (NRC 1980)
      1. Sheep, 25 ppm; 10-15 ppm can be toxic to sheep if diets contain only 0.1 ppm Mo or if concentrates are fed for a prolonged period
      2. Cattle, 50-100 ppm; 50 ppm may be toxic to calves. Bovines are more resistant to Cu poisoning than sheep but are also more susceptible to Cu deficiency
   4. There are three relatively distinct stages in development of Cu toxicity in ruminants
      1. Gradual accumulation of Cu in the tissues, particularly the liver
         1. Blood Cu values are normal
         2. No clinical symptoms are evident
      2. Whole blood Cu may rise to twice normal levels
         1. Plasma bilirublin may increase
         2. Decreased liver function
         3. Increases in a variety of blood enzymes, plasma glutamic oxaloacetic transminase (PGOT), arginase
      3. The hemolytic crisis
         1. Whole blood Cu increases to 5-8 X normal
         2. Total hemoglobin drops but methemoglobin increases
         3. Increased oxidative state of blood
         4. Rise in serum creatine phosphokinase
         5. Erythrocyte distortion
         6. Blood glutathione concentration falls drastically
   5. Clinical symptoms
      1. Dullness
      2. Anorexia
      3. Dehydration
      4. Acute thirst
      5. Evidence of abdominal pain
      6. Jaundice
      7. Hemoglobinurea
   6. Postmortem findings
      1. Generalized jaundice, adipose tissue yellow
      2. Kidney has a distinctive black metallic sheen
      3. Urinary bladder and gall bladder are apt to be distended with dark-colored fluids
      4. Spleen is enlarged, soft and dark
   7. Histological changes
      1. Fatty liver
      2. Dilation and necrosis of renal tubules
      3. Petechial hemorrhages on the heart
      4. Splenic hemosiderosis
   8. Hemolytic crisis can be triggered by stress, change of environment, transportation, handling, etc.
2. Causes of Cu poisoning in ruminants
   1. Excessive Cu doses as anthelmintics
   2. Excessive Cu doses to correct Cu deficiency
   3. Accidental consumption of high Cu feed intended for swine
   4. Grazing too soon after application of Cu-containing fertilizer or high Cu swine or poultry manure to pasture
   5. Exposure to Cu-based fungicides or insecticides
   6. Hepatoxic plants may make the liver more susceptible to accumulation of Cu
   7. Prolonged feeding of concentrates to confined sheep can be dangerous, particularly if dietary Mo and S are low
3. Treatment or prevention of Cu poisoning in ruminants
   1. Supplementation of feed with 0.1 g ammonium molybdate + I g sodium sulfate/sheep/day to reduce liver Cu
   2. IV administration of thiomolybdate to reduce liver Cu
   3. Dietary Zn 5-10X requirement may have a protective effect against excessive dietary Cu

Copper — Cu (atomic number 29)

A trace metal in enzymes that handle oxygen and make connective tissue. Balance with zinc matters: high zinc for a long time can drive copper down.

Vitamins, Major Elements, Trace Elements, Toxic Elements

Vitamins

Vitamin A	Vitamin B1 (Thiamin)	Vitamin B2 (Riboflavin)	Vitamin B3 (Niacin)
Vitamin B5 (Pantothenic Acid)	Vitamin B6	Vitamin B12	Vitamin C
Vitamin D	Vitamin E	Vitamin K	Biotin
Folic Acid

Major Elements

Calcium	Magnesium	Phosphorus	Sodium, Potassium, Chloride
Sulfur (Sulfer)

Trace Elements

Boron	Chromium	Cobalt	Copper
Fluorine	Iodine	Iron	Manganese
Molybdenum	Nickel (Nickle)	Selenium	Silicon
Tin	Vanadium	Zinc	Ultra Trace

Toxic Elements

Aluminum	Arsenic	Cadmium	Lead
Mercury