Nutritional Assessment of Minerals

1. Nutritional Assessment of Minerals How much is too much How much is not enough How can we tell the difference

2. NUTRITIONAL CONCERNS Evaluating Individual Need Evaluating Individual Status Setting Standards for optima Population Approach Experimental Biomarkers Balance Studies Functional tests

3. Traditional Experimental Approaches to Determine Optimal Levels Balance Studies Purified Diets with varying Mineral Content Standards of Excellence

4. Kout Kin A B D K1 K-1 Retention C Kin Kout = Balance Kin Kout > Positive Balance (growth) Absorption-Excretion Negative Balance (wasting) Kin Kout < K1 = K-1 Balance K1 K-1 Retention > Positive Balance K1 < K-1 Negative Balance Balance Matching what goes in with what goes out Absorption Excretion

5. Problems With Balance as a Criterion of Amount 1. End Point is in doubt 2. Excretion is episodic not continuous 3. Multiple connecting pools of the same mineral 4. System adaptation

6. Experimental and Clinical Approaches 1. Experimental generally applicable to animal studies a. Semi Purified diet approach b. Make quantity of mineral in diet limiting c. Measure rate of growth, changes in a biomarker d. Assess quantity needed to suppress symptoms e. Assess quantity needed to reverse symptoms 2. Clinical approaches generally applicable to humans a. Correlate symptoms with diet record of patient b. Determine level needed to suppress or reverse pathologies

7. Standards of Excellence Correlate amount needed with a high quality food source Example: Human or Bovine Milk to determine mineral levels for optimal health Problems: 1. Milk is poor in iron and copper 2. Milk contains whey protein that blocks calcium absorption

8. Population Based Studies* Correlate level with risk Assign values based on a healthy population Assign ranges for adequate, subadequate, and toxic levels *Population based studies are risk assessments that rely on statistics and Gausian Distribution

9. Traditional Approaches to Determining the Mineral Status of an Individual 1. Body stores of the mineral 2. Overt response to increased mineral intake 3. Physical signs of a deficiency a. Stunted growth b. Overt abnormalities 4. Internal signs of a deficiency - biomarkers a. Tissue or blood levels b. Mineral binding proteins levels 5. Functional Assays a. Enzyme assays

10. Biomarkers of Nutritional Adequacy intestine Dynamic storage Plasma Rapid turnover Functional pool Excretion Slow turnover

11. Selenium A good mineral to assess

12. H H CH3-S-CH2CH2C-COOH CH3-Se-CH2CH2C-COOH NH3 L-Methionine NH3 Selenomethionine (major dietary source) Plasma Selenium (8-10 g/L) • Glutathione peroxidase (GPX3) • Selenoprotein P (plasma), W (white muscle disease) + + Most selenium absorbed goes into muscle and is not under homeostatic control But, plasma selenium rises on selenium intake

13. OTHER BIOMARKERS OF Se Whole blood, hair and nail (applies to chronological intake) Example: People in China where Keshan’s disease is prevalent were assessed for selenium status by hair and nail measurements of selenium Plasma Glutathione peroxidase (GPX3) activity. Advantages 1. Can readily spot values in the deficient range 2. Concern for contamination is minimal 3. Can be done by a rapid automated procedure Disadvantages 1. Values plateau when intake exceeds optimum 2. Enzyme unstable to storage 3. Laboratory to laboratory variations 4. GPX3 activity is affected by other nutrient deficiencies

14. Conclusions The practical minimum requirement for Se is that which prevents Keshan’s disease Recommended intakes of Se have been calculated from the requirement for optimal glutathione peroxidase (GPX3) activity and biologically necessary Se compounds Because GPX3 activity plateaus with increasing Se, its difficult to ascribe a particular level of Se adequacy based on this index Suboptimal or marginal Se intake may be monitored by GPX3 mRNA, but this requires further study

15. Ca2+ Its all about bone Biomarkers Bone Resorption urinary deoxypyridinoline (DPD) collagen-type 1 N-telopeptide (NTX) Other markers of calcium-related bone Formation serum osteocalcin (OC) bone-specific alkaline phosphatase (Bone ALP)

16. Ca2+ COOH HOOC COOH CH CH2 CH2 CH2 CH C CH N C N O H O H Osteocalcin HA ( a small 49 residue Ca2+-binding protein that binds tightly to hydroxyapatite) Vitamin K GLA (carboxy glutamic acid) Osteocalcin makes up about 10-20% of the non-collagenous protein in bone. Synthesized by osteoblasts it functions in calcium homeostasis, binding tightly to hydroxyapatite mineral surfaces. Serum levels of osteocalcin have been correlated with bone mineral density.

17. Copper Acceptable but in need of repair

18. Currently Accepted Biomarkers Serum copper Serum ceruloplasmin Biomarkers to Consider Erythrocyte Cu/Zn superoxide dismutase (severe only) Platelet or lymphocyte cytochrome c oxidase Biomarkers on the Horizon PAM (peptidylamide monooxygenase) Copper chaperone for Superoxide dismutase

19. Problems with Copper Biomarkers 1. Ceruloplasmin is an acute phase protein elevated by: Infections Inflammations stress 2. Ceruloplasmin levels rise in response to estrogen. Depression of ceruloplasmin and serum Cu may not be evident 3. Ceruloplasmin synthesis my be suppressed by protein deficiency 4. Serum Cu and ceruloplasmin levels are age dependent Neonates and children are normally lower than adults

20. Experimental Data Feeding postmenopausal women a diet of 0.57 mg Cu/day for 3 months did not depress plasma Cu or ceruloplasmin. Platelet and leukocyte cytochrome c oxidase activity, however, was suppressed. But feeding adult men 0.38 mg Cu/day for 6 weeks did depress serum Cu and ceruloplasmin. (Milne and Nielsen Am. J. Clin. Nutr. 63, 358-364, 1996) Healthy adult volunteers fed 50-60 ug additional Cu/kg/day for 3 months failed to show any rise in superoxide dismutase activity or serum ceruloplasmin. These concentrations failed to change the range of Cu homeostasis. (Araya et al. Biometals 16, 199-204, 2003.

21. Peptidylglycine-alpha amidating monooxygenase (PAM) Active hormone O Cu O H H PAM C C C H Pep N Pep N COOH H H + HOOC-CHO glyoxylate

22. Galanin (monoaminergic neurons) Gastrin (gastric acid) Vasopressin (water homeostasis) Thyrotropin releasing horomone (thyroid hormone) Neuropeptide Y (hunger, obesity) Pancreastatin (Insulin control) Calcitonin (osteoporosis) Gonadotropin releasing hormone (sex hormones) Substance P (emotions) PAM Cu

23. Experimental data Mice with a genetic defect in ATP7A, a membrane Cu transport protein, were observed to have depressed levels of 5 of 6 pituitary peptides identified with PAM activity. All the peptides had the C-terminal amidated group. (Chei et al, Cell Mol. Biol (Noisy-le-grand) 49, 713-722, 2003) Liver and erythrocyte copper chaperone for Cu/Zn superoxide dismutase (CCS) was significantly increased in rats fed a high Zn diet as compared to low Zn. CCS is a sensitive measure of Zn-induced Cu deficiency. (Iskandar et al, Nutr. J. 4, 35, 2005)

24. Mn2+ Not a clinical or public health concern Plasma levels reflect dietary levels: down with restriction, up with supplements

25. Molybdenum Biomarkers for deficiency 1. Decrease urinary sulfate and uric acid 2. Elevated sulfite, hypoxanthine and xanthine Adenine Hypoxanthine Xanthine oxidase a Mo2+ enzyme Xanthine Uric Acid

26. Chromium A rough History Biomarkers of Deficiency Impaired glucose tolerance Plasma not a good indicator Urinary excretion controversial

27. Acceptable range of intake LOAEL EAR RNI UL % at Risk Cumulative Risk 1.0 Risk of deficit 0.5 Risk of excess NOAEL 50% 2.5% 0% Intake

28. Safety Cumulative Risk 1.0 0.5 RNI UL EAR Intake