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!! !! !! !!

Overview! Food Sources of Copper! DRI! Functions!

"! "!

Jennifer Messana-Eaton! NFSC 642! Spring 2013!

"!
!! !! !! !! !! !! !!

Enzymatic! Non-Enzymatic! Copper & cardiovascular health!

Associated deficiencies! Nutrient Interactions! Toxicity! Supplement Sources! Assessing copper status! Research Article! Follow-Up Study!

!! In
!!

the body !
in either of two valence states!

!! !!

Varies widely! Richest sources:!

"! "!

"! Found

Cuprous state (Cu1+)! !! Cupric state (Cu2+)!

!! Approximate !! Essential!
"! Enzyme

organ meats! shellfish! Nuts & seeds! Legumes! Dried fruits! potatoes! whole grains! Cocoa! GI tract! !! Secreted daily into the GI tract in digestive juices, in relatively large amounts!

!!

Plant sources:!
"! "! "!

body content, 50-150 mg!

!!

Good sources: !
"! "! "!

cofactor! "! Allosteric component of enzymes! "! Intermediate in electron transfer!

!!

Endogenous sources: !
"!

!! Median !! RDA!
"! Adult
!!

intake!
1.0 to 1.6 mg/day!

"! Adults

men & women!

0.9 mg/day!

!! Pregnant
"! 1.0 "! 1.3

women! women!

mg/day! mg/day!

!! Lactating

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!! Enzymatic! "! Ceruloplasmin! "! Lysine oxidase! "! Tyrosine Metabolism/Catabolism! "! Superoxide dismutase! "! Cytochrome c oxidase! "! Amine oxidases! "! Amination of peptides! !! Nonenzymatic! !! Copper

!!
"! "! "!

Ceruloplasmin (ferroxidase I):!

Glycoprotein! Transports copper in blood! Oxidative enzyme!
!!

Oxidizes Iron so that it can be transported to tissues! #! Iron must be oxidized to bind to transferrin!

& cardiovascular health!

"!

Associated deficiency!
!!

Secondary Iron deficiency anemia!

!!
"!

Ceruloplasmin: Continued!
Antioxidant!
!! !! !!

!!
"! "!

Lysine Oxidase: (Lysyl Oxidase)!

Secreted by connective tissue! Generates cross-links!
!!

In plasma! Bound to cell surface receptors on plasma membrane! Acute phase protein! #! Modulator of inflammatory process! #! Scavanger of free radicals!
"!

#!

Between connective tissue proteins! Including collagen & elastin! Catalyzes removal of Amino group! From lysyl & hydroxylysyl residues ! Oxidation of the terminal carbon forming crosslinks! Cross-linking is needed to stabilize the extracellular matrix!

"!
!!

Involved in Oxidative Deamination!

#! #! #!

Phagocytes generate superoxide radicals!

"!

Associated deficiency!
!!

Hyperchromic anemia! #! Tiny, pale RBC!

"!

Never reached sufficient level of Hgb!

!!
"!

Lysine Oxidase: continued!

decreases with inadequate Copper intake!
!!

!! Tyrosine
!!

Metabolism!
of Norepinephrine/Melanin!

"! Synthesis

Negatively affects connective tissue!

"!
!!

Associated deficiency!
Cardiac & Skeletal defects!

Copper dependent! #! Tyrosine --# L-dopa! #! L-dopa -# Dopamine!

"!

Dopamine monooxygenase converts dopamine to NE! #! Contains up to eight copper atoms per molecule! #! Requires O2 & Vitamin C!

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!! Tyrosine
melanin

Catabolism!

"! Copper

& Vitamin C dependent! !! Conversion of p-hydroxyphenylpyruvate to homogentisate!

"! Associated
!!

deficiency!

Altered skin pigmentation!

Fig. 6-28, p. 213

!! Superoxide
melanin

Dismutase (SOD)!

Imidiazole

"! Cytosol
!!

"! Antioxidant

& extracellular! defense!

Histidine

Associated deficiency! #! Cardiovascular & pulmonary dysfunction!

"! Copper
!! !!

& zinc dependent!

Linked through Imidiazole group! Both linked to Histidine & Aspartate residues! !! Copper is located at the active site!
Fig. 6-28, p. 213

!!

SOD: continued!
"! "!

Catalytic function! Rids body of superoxide radicals !

!!

Copper is reduced!

!! Cytochrome c Oxidase! "! Contains 3 copper atoms/molecule! "! Functions!

!! !! !!

"! "!

SOD serves a protective function!

!!

Electron transfer! Reducing O2! Mitochondrial electron transport (ETC)! #! Transfers electron to reduce O2! #! Form water!
"!

Superoxide radicals lead to hydroxyl radicals! Decreased enzyme activity! #! Increased peroxidation of cell membranes

Generate ATP!

Associated deficiency!
!!

"!

Associated deficiency!
!!

Impaired enzyme activity!

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!! Amine
!!

Oxidases!
dependent!

!! Amidation
"! Copper
!!

of peptides!

"! Copper

"! Horomone

Allosteric structural component! !! Histidine serves as ligands!

"! Found
!!

fucntion! dependent enzyme!

in blood & Body tissue!

Catalyze oxidation of biogenic amines! #! Tyramine, Histamine, Serotonin, Polyamines! #! Form aldehydes & ammonium ions! !! O2 reduced # H2O2!

Peptidylglycine alpha-amidating monooxygenase! #! Located in the brain! !! Cleaves carboxy terminal glycine residue! !! Off peptides ! #! C-terminal glycine! !! Requires vitamin c! #! Reduces cu2+ back to cu1+!

!! Coppers
"!
!!

other roles !

!! Coppers
"! Studies
!!

role in CVD!
show a role!

Not well understood!

Angiogenesis! #! Development of new blood vessels! Immune system function! Nerve myelination! #! Central nervous system! Endorphin action! Differentiation of neutrophils! Leukopenia, neutropenia!

"! Antioxidant!

!! !!

!! !!

Positive/negative correlation! !! Correlation between Copper & atherosclerosis! !! CVD system sensitive to Copper deficiency! !! Too high or too low possibly detrimental!

"!

Associated deficiencies!
!!

!!

Deficiency in copper!
"! "! "! "! "! "! "!

!! !!

Vitamin C!
"! "!

Hyperchromic anemia! Secondary iron deficiency anemia!

!!

Reduces levels of copper! Mutual antagonist!

!! !!

Treatment with copper & not iron!

Zinc!
Excessive intracellular binding of copper! Reducing entry into the blood!

Leukopenia! Hypopigmentation! Bone abnormalities! Impaired immune function! Cardiovascular & pulmonary dysfunction! Nephrosis!
!!

!!

Iron!
"! "!

Ceruloplasmins role!
!!

Oxidizing iron! Interferes with copper!

!!

Diseases which promote copper loss!

"! "!

High levels of iron!

!!

Kidney disease! Celiac disease & inflammatory bowel disease !

!!

Selenium!
"!

Gastrointestinal maladaptive disorders!

!!

Reduced glutathione peroxidase!

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!! UL!
"! 10
!!

!! Fortified
"! Copper

foods!

mg/day! "! >64 mg/day!

Epigastric pain, nausea, vomiting & diarrhea! !! Hematuria, liver & kidney damage!
"! 1,000
!!

sulfate! oxide!

!! Supplements!
"! Cupric
!!

X normal!
!!

Discouraged! #! Un-absorbable in GI of animals!

lethal!

"! Copper

Glycinate!

Better absorbtion!

!!

Multiple indicators!
"! "!

Serum, plasma, RBC copper!

!!

Inadequate short term changes! Unrelated to diet! 10 micromol/L! < 0.38 mg/day! 180 mg/L! < 20 mg/L! 0.47 0.067 mg/g)! Sensitive to long-term status!

Affected by several factors!

!! !! !!

"!

Ceruloplasmin!
!! !!

R.A. DiSilvestro, E.L. Joseph, W. Zhang, A.E. Raimo, Y. Min Kim Metabolism clinical & experimental.2012; 61, 1242-1246!

"!

Superoxide Dismutase!
!! !!

!! To

determine the effect of copper supplementation on blood copper enzyme activities & the parameters related to cardiovascular health! !! Hypothesis!
"! Blood

!! Independent
"! Dose

Variable!

of Copper!

!! Dependent
"! Blood

variables!

Copper Enzymes! "! Markers of CVD risk!

copper enzyme activities, as well as blood markers of cardiovascular health, would respond to copper supplementation!

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!! Confounding "! None Listed!

Variables!

!!

Adult males & females!

"!

N=35/group!
!! !!

!! Should be included?! "! Diet! "! Exercise! "! Ethnicity! "! Hypertension! "! Liver/Kidney disease! "! History of Heart disease! "! Family history!

23 females! 12 males! 8 weeks! Before & after! Primary measures! #! Blood copper enzyme activities! Secondary measures! #! Blood parameters relevant to Cardiovascular health! Maintain current!

"! "!

Time!
!!

Blood samples!
!! !!

!!

"!

Diet & exercise!

!!

!! Inclusion Criteria! "! 45-60 years of age! "! post-menopausal! "! Plasma cholesterol level!
!!

!! Randomized
"! Placebo

control trial!

controlled! "! Double blind!

Copper 2 mg Copper Glycinate Same size & appearance Placebo Maltodextrin

Between 190 - 270 mg/dl!

!! Exclusion
!! !! !!

criteria!

Major health problems! Medications affecting copper metabolism! Supplements with copper! #! Multi vitamins with < 2 mg allowed! #! Discontinued for study!

!!

Laboratory analysis!
"! "!

Pre-study!
!!

!! Paired

t-test!

Cholesterol levels! Pre! Post! Plasma ceruloplasmin! Superoxide Dismutase! Measured! #! Oxidized ldl! #! C-reactive protein! #! homocystein!

Blood collected!
!! !!

"! P-value "! P-value

"! "! "!

Plasma!
!!

< .05 Ceruloplasmin! < .01 Superoxide Dismutase! "! Compared pre & post values!
!! Pearson
"! P-value

Erythrocytes!
!!

Correlation Coefficients!
< .05!

Elisa kits!
!!

!! All
"! Ex

analyses!
Vivo!

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!! Ceruloplasmin
"! "! "! "!

Activity!

!! Erythrocyte
"! "! "! "!

Superoxide Dismutase activity!

Figure 1! Paired t-test!

!!

Figure 2! Paired t-test!

!!

P-value < .05!

P-value < .01!

Pre/Post supplementation! Ex vivo!

!!

Pre/Post supplementation! Ex vivo!

!!

!!

Copper values ! #! Significant change! Placebo! #! No change!

!!

Copper values! #! Significant change! Placebo! #! No change!

!! Cholesterol
"! "! "!

measures!

!!

SOD & 5 Cardiovascular values!

"! "!

Table 1! Paired t-test!

!!

Pearson Correlation Coefficient!

!!

P-value < .05! Positive Correlation (somewhat)! Placebo: 0.58 > .05 p-value! Copper: 0.38 > .05 p-value! Negative correlation! Placebo: -0.57 > .05 p-value! Copper: -0.48 > .05 p-value!

P-value < .05! No significant changes! #! Total Cholesterol! #! LDL! #! HDL! #! C-Reactive Protein! #! Homocystein!

SOD & HDL!

!! !! !!

Pre & Post supplementation!

!!

"!

SOD & Homocystein!

!! !! !!

!! Oxidized
"! "! "!

LDL!

!!

Figure 3! Paired t-test!

!!

P-value < .05! Copper values! #! Decreased significantly! #! Inconsistent changes!

"!

Pre/post supplementation!
!!

In middle aged adults with moderately high cholesterol, copper supplementation raised the activities of two blood copper enzymes. Relevance to these results are not yet established, more research is necessary to better understand implications.!

21 of 35 subjects !

!!

Placebo! #! No change!

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!! RCT! !! Pre

!! !! !! !! !!

Ex vivo! Ethnicity! diet analysis! Physical activity! Population studied!

"!

supplementation values!
same before trial!

"! Groups

Generalizability! Copper < 2mg/day!

!! Exclusion

criteria! !! Copper glycinate! !! Statistical analyses! !! assessment!

!!

Supplements!
"!

!! !! !! !!

Measurement of Compliance! Time! Sample size! Randomization!

"!

Uneven grouping between gender!

!!

Statistical Analysis!

!!

What were the specifics of the authors recuritment?!

"! "!

How many subjects did they start out with?! Were their any drop outs?! Gender! Copper! placebo! Did they run an analysis and not include the results?!

!!

Purpose!
"!

!!

What were the within group comparisons?!

"! "! "! "!

To investigate the association between copper status and risk of cardiovascular disease! Prospective Cohort Study! Adults 45-65 years of age! 5,000 subjects! Healthy individuals! Independent: Copper Status! Dependent: Risk of CVD!

!! !!

Study design!
"! "! "! "!

Subjects!

!!

Did they run a pearson correlation coefficient on ceruloplasmin & 5 cholesterol measures?!
"!

What were the results?! Were the subjects considered deficient?!

!!

What were the Pre values for copper status?!

"!

!!

Variables!
"! "!

!!

Would their be a correlation between blood copper enzyme values and 5 CVD risk markers in free living people?!

!!

Methods!
"! "! "!

!!

Patients followed over 10 years! Assessed at baseline & every 6 months for 10 years! Questionnaire!
!!

Diet, physical activity, medical history, chronic diseases, Family history, socioeconomic status, ethnicity, supplements!
!!

DiSilvestro, Robert A., Elizabeth L. Joseph, Wenyi Zhang, Adrienne E. Raimo, and Young Min Kim. "A Randomized Trial of Copper Supplementation Effects on Blood Copper Enzyme Activities and Parameters Related to Cardiovascular Health." Metabolism Clinical & Experimental 61 (2012): 1242-246. Print.! Gropper, Sareen Annora Stepnick., Jack L. Smith, and James L. Groff. Advanced Nutrition and Human Metabolism. Australia: Wadsworth/ Cengage Learning, 2009. Print. ! Hansen, S. L., P. Schlegel, L. R. Legleiter, K. E. Lloyd, and J. W. Spears. "Bioavailability of Copper from Copper Glycinate in Steers Fed High Dietary Sulfur and Molybdenum." Journal of Animal Science 86.1 (2007): 173-79. Print.! Kang, James Y. "Copper and Homocystein in Cardiovascular Diseases." Pharmacology & Therapeutics 129 (2011): 321-31. Print.!

"!

Blood Samples!
!!

Ceruloplasmin, Sod, oxidized LDL, Total Cholesterol, HDL, LDL, C-reactive Protein, Homocystein! Paired t-test! #! To compare copper values to CVD risk factors & within group analysis ! Pearson correlation coefficient! #! To test the associations between the copper copper status & CVD risk factors and age! #! To test the associations between the copper copper status & CVD risk factors and gender! Linear regression analysis! #! To see if copper status predicts CVD changes in the subjects!
!!

!!

Statistical Analysis!
!! !!

!!

!!

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