Micronutrient Undernutrition in Americans Nutrient Population Group % ingesting % ingesting RDA Minerals Iron Women 20-30 years 18 mg 75% 25% Women 50+ years 8 mg 25% 5-10% Zinc Men; Women 50+ years 11; 8 mg 50% 10% Vitamins B6 Men; Women 1.7; 1.5 mg 50% 10% Folate** Men; Women 400 mcg 75% 25%; 50% B12 Men; Women 2.4 mcg 10-20; 25-50 % 5; ~10-25% C Men; Women 90; 75 mg 50% 25% •Wakimoto and Block (2001) J Gerontol A Biol Sci Med Sci. Oct; 56 Spec No 2(2):65-80. ** Before U.S. Food Fortification Methionine Serine B6 CH3-THF B12 SHMT MTHFR (polymorphism) MS CH2=THF TS Homocysteine dUMP dTMP Micronuclei in: RNA positive reticulocytes RNA negative erythrocytes 130 Folinic Acid Folic Acid 80 40 30 20 Micronuclei per 1000 cells Micronuclei per Normal 0 range 1 year 50 100 150 200 250 300 350 preRx TIME (DAYS) Uracil/10,000 Micronuclei thymine (mn reticulocytes/1000 reticulocytes) 100 100 10 10 1 <140 175-200 240-300 >300 <140 175-200 240-300 >300 Erythrocyte Folate (ng/ml) Erythrocyte Folate (ng/ml) 10% US population Presupplementation 50% Low income minority Postsupplementation 60 50 40 30 20 MIN PCEs/1000 PCEs MIN PCEs/1000 x 10 x 0 0 5 10 15 20 25 30 PLASMA FOLATE (NG/ML) Mean uracil content in Folate Deficiency Study sperm DNA from 23 men epidermal Sperm Count on diets low in fruits and vegetables DC uracil 14 100 in DNA Epidermal 12 Sperm 10 8 50 6 4 0 2 0 bna_ 0011_009.ppt Seminal plasma folates vs. semen quality Correlation coefficient (r); n=48 Seminal Plasma Sperm Density (106/mL) Total Sperm Count (105) Non-methyl THF * 0.37 * 0.31 (methylene-THF, etc.) 5-Methyl THF 0.08 0.07 *p<0.05 Human Lymphocyte DNA Strand Breaks (Comet Assay) vs. B-6 Intake 3.5 3.0 ZE-1 2.5 TK-2 AW-4 2.0 TB-5 MR-6 LS-7 100 comets 100 1.5 AB-8 CH-9 Avg 1.0 RB-12 YHC-13 HP-16 0.5 TS-10 0.0 Baseline 0.7 1.4 2.2 10.3 Vitamin B-6 Intake (mg/d) T. Shultz, C. Hansen, K. Hunt, J. Leklem, A. Huang, & B. Ames Human Lymphocyte DNA Strand Breaks (Comet Assay) vs. B-6 Intake 3.5 3 2.5 2 1.5 1 0.5 0 024681012 Vitamin B-6 Intake Analysis of nonlinear regression models: comparison of an overall model and individual models of Z-transformed values vs. ln- nonheme liver iron 3 2.5 Over all 2 DCF-PMNs 1.5 DCF-Lymph 1 Rh123-PMNs Rh123-Lymph 0.5 Z score mtDNA damage 0 1/RCR -0.5 -1 normal -1.5 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 LN nonheme Fe (µmol/g wet liver) . Each of the six dependent variables (that were analyzed by nonlinear regression in former figures) were transformed to Z scores and modeled as a quadratic function of the ln-liver nonheme iron as the independent variable. The equation for the RCR ratio's Z score was obtained from inverted RCR values (1/RCR) so that normal rats had the lower instead of the higher values. For presentation purposes each model line was obtained from 9 values of liver iron. All statistics were performed as in materials and methods. Synthesis of Heme Cytosol Other intermediates PBG 2ALA PPIX PPGIX FeII FC Mitochondria protoheme Succ-CoA + Gly ALA Loss of activity of complex IV in heme-deficient cells 2.5 2 1.5 1 0.5 *** *** 0 Control 5µM NMP 10µM NMP Heme deficiency induces oxidative stress Similarity Between the Consequences of Heme Deficiency and Normal Aging/neurodegeneration Factor in Study Heme Deficiency Aging/Neurodegeneration Complex IV Loss of complex IV 9 Loss of complex IV Iron Accumulation 11 Accumulation Oxidative Stress Increased 9 Increased APP Decreased and dimmer or aggregate aggregate appear 11 NOS Increased 11 Increased Cell-cycle and Disabled differentiation Loss of Axons; differentiation or proliferation 11 neuronal death Metabolism Mitochondrial decline 9,10 Hypometabolism Calcium Corrupted 9 Corrupted Ferrochelatase Increased 9 Increased in senescent cells 9* Heme synthesis Decreased 10 Decreased with age** *Not Determined in vivo. **Not determined in the aging brain 9) Atamna et al (2001) JBC. 10) Atamna et al (2002) ABB. 11) Atamna et al (2002) PNAS. Zinc Deficiency Induces Fapy Glycosylase (Fpg)-sensitive Single Strand Breaks in Human Lung Fibroblasts Control (+Fpg) ZnAD (+Fpg) ZnDF (+Fpg) 200 * 160 120 80 40 Comet Score 0 Control ZnAD ZnDF ACKNOWLEDGEMENTS Children’s Hospital Oakland Research Institute University of California at Berkeley Dr. Hani Atamna Ms. Susan Mashiyama Dr. Lois Gold Dr. Lynn Wallock Mr. Tom Slone Dr. Patrick Walter Dr. Neela Manley Dr. Arnold Huang Dr. Chantal Courtemanche 35 Dr. Emily Ho High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme-binding affinity (increased Km): Relevance to genetic disease and polymorphisms Bruce N Ames, Ilan Elson-Schwab, and Eli A Silver, Am J Clin Nutr 2002; 75:616-658 Summary of Work Many genetic diseases in humans are ameliorated by the administration of high levels of vitamins. An appreciable percentage of mutations in a gene, perhaps a third, results in the corresponding enzyme having an increased Km (poorer binding affinity) for a coenzyme or substrate, resulting in a lower rate of reaction. Because the intracellular concentration of coenzyme can often be increased therapeutically, enzymatic activity can be restored at least partially, and the disease phenotype cured or ameliorated. We have documented about 50 human genetic diseases involving dysfunctional enzymes which can be remedied or ameliorated by high levels of the vitamin component of the coenzyme, and a number of other genetic diseases, including some due to polymorphisms, where this approach may be useful. Mitochondrial Ornithine Amino Transferase & Gyrate Atrophy of the Choroid and Retina • Defective OAT leads to accumulation of ornithine and sight degradation. • The Km of OAT for PLP (B6 cofactor) is increased (7 - 20x) in ~5% of patients. • B6 therapy lowers ornithine levels. Genetic disorders affect micronutrient sufficiency of the following vitamins and nutrients 1. Pyridoxine (Vitamin B6) 11. Tetrahydrobiopterin 2. Thiamine (Vitamin B1) 12. S-Adenosyl Methionine 3. Riboflavin (Vitamin B2) 13. Pantothenic Acid 4. Niacin (Vitamin B3) 14. Lipoic Acid 5. Biotin (Vitamin B7) 15. Carnitine 6. Cobalamin (Vitamin B12) 16. Hormones 7. Folic Acid 17. Amino Acids 8. Vitamin K 18. Metals 9. Vitamin D 19. Maxi B Vitamins 10. Vitamin E A number of polymorphisms affect coenzyme binding and may be remediable by high-dose vitamins • Methylenetetrahydrofolate Reductase C677T and FAD • Glucose-6-Phosphate Dehydrogenase A44G and NADP • NAD(P):Quinone Oxidoreductase 1 (DT-diaphorase) P187S and FAD • Aldehyde Dehydrogenase E487K and NAD • Short-Chain Acyl-CoA Dehydrogenase G209S and FAD + + Cellular Cytoplasm H H + H+ H H+ H+ MMitochondrialitochondrial Outer Membrane Intermembrane H+ + H + Space H+ H H+ H+ H+ H+ H+ IInnernner Membrane II CoQCoQ IIIIII CytCCytC NADH IIII VV Mitochondrial + IVIV + H Matrix NAD FAD H+ FADH O2 + 2 ADP H H2O + Succinate H+ ATP H Fumarate ATP H+ Succinyl-Co-A CITRIC α-Ketoglutarate NADH L-Malate ACID Dehydrogenase NADH CYCLE Complex Pyruvate NADH α-Ketoglutarate + Dehydrogenase Oxaloacetate H H+ complex NADH Acetyl-Co-A Citrate Isocitrate Citrate 9 Synthase Effect of ALCAR Supplementation on Cardiolipin Levels 30 20 g per 10 cells) µ ( + ALCAR 10 Cardiolipin 0 Young Old 14 YOUNG NO R123 Fluorescence ALCAR in Young and Old WITH Rat Hepatocytes ALCAR Normalized Cell Number Cell Normalized NO OLD ALCAR WITH ALCAR Normalized Cell Number Cell Normalized Lipoic Acid Lowers Mitochondrial Oxidants in Old Rats 20 ** nute i 10 Consumed per M Consumed per 2 + LA + LA Fl. Units/O 0 Young Old 17 Age-associated decrease in immune function and the effect of ALCAR (0.2%) + LA (0.1%) treatement for 2 months. Values are mean + SEM of 10-11 animals. P<0.001 30 20 p<0.01 10 T cell stimulation index 0 ed t Old ted ea a Young Old Tre Young Tr MDA levels in young and old rats with LA, ALCAR, or both 80 P<0.05 70 ***p<0.001 vs. young rat group P<0.01 60 50 Young Old *** 40 30 MDA (pmol/mg protein) (pmol/mg MDA 20 10 0 + LA + LA + ALCAR + ALCAR + ALCAR + LA + ALCAR + LA 20 Ambulatory Activity before and After Supplementation with Lipoic Acid (LA) + Acetyl-L-Carnitine (ALCAR) 800 * * vs. young # vs. old 600 # 400 * 200 + LA + ALCAR Distance Travelled (cm/hour/day) Travelled Distance + LA + ALCAR 0 Young Old Morris Water Maze for Testing Spatial Memory Spatial Memory relies on intact hippocampal function. Treatments improved poor memory in old rats 22 Spatial Memory Tested With Morris Water Maze 100 P<0.001 P<0.05 80 60 40 + LA + ALCAR 20 + ALCAR + LA 0 Young Old Old old Old 23 Peak procedure: for measuring temporal memory. Associated with striatum, cerebellum, & hippocampus 14 PEAK RATE: 12.00 measures learning Young 12 and motivation. 10.00 Old Old + ALCAR 10 PEAK TIME: measures Old + LA internal clock, food is 8.00 Old + ALCAR + LA 8 rewarded only when animals push lever 40s 6.00 6 after sound or light signal 4.00 4.00 2.00 2.00 0.00 0.00 0 50 100 150 200 0 50 100 150 200 SOUND: Time to Signal LIGHT: Time to Signal 25 Oxidative Damage to Nucleic Acid in Old Rats by mAb to oxo8G/oxo8dG: Immunohistochemical stain of neurons 26 Staining of oxidized nucleic acid in neurons (mAb to oxo8dG in DNA/oxo8G in R NA) RNA is Oxidized (92% is removed by RNase) *oxo8G: 8-hydroxyguanosine; oxo8dG: 8-hydroxy-2’-deoxyguanosine 27 Dendritic spine density of cortical neurons in rat brain with Golgi staining: Effects of 11 month treatment with ALCAR, LA or ALCAR+LA ** * 70 60 50 40 30 Spine density 20 10 0 Young Old +ALCAR +LA +ALCAR +LA Liu, Mervis, and Ames, Unpublished data 10 10 1/v 1/v 5 5 0 0 -10 0 10 20 30 40 -50 0 50 100 150 200 1/[ALCAR, mM] 1/[CoA, mM] 1200 ** young old = vs. young 150 * # = vs. old # # 800 * # # 100 M µ 400 ** nmol/min/g 50 # 0 0 Vmax Km for Km for ALCAR CoA 0.8 A 0.6 MDA 0 /min] MDA 12.5 µM 340 0.4 MDA 25 µM MDA 50 µM 0.2 MDA 100 µM 1/v [mOD 0 0 0.005 0.01 0.125 0.1 B HNE 0 /min] 0.075 HNE 500 µM 340 HNE 1000 µM 0.05 HNE 2000 µM 0.025 1/v [mOD 0 -0.005 0 0.005 0.01 0.15 1/S [ALCAR µM] CC Control MDA (50 µM) 0.1 MDA+ALCAR /min] MDA+L-carnitine 340 MDA+CoA 0.05 MDA+Acetyl-CoACoA 1/v [mOD 0 -0.005 0 0.005 0.01 29 1/S [ALCAR uM] ACKNOWLEDGEMENTS Children’s Hospital Oakland Research Institute University of California at Berkeley University of California at Irvine Linus Pauling Institute, Oregon State University Dr. Jiankang Liu Dr. Tory Hagen Prof. Fernando Viteri Dr. Afshin Gharib Dr. Mitch Knutson Dr. David Killilea Dr. Elizabeth Head Dr. Patrick Walter Dr. Carl W. Cotman Dr. Hani Atamna 35 Dr. Emily Ho