~30% Smoking ~35% Unbalanced Diets Too Many Calories: Obesity Too Little Fiber & Micronutrients ~20% Chronic Infections Mostly in Poor Countries ~20% Hormones.

~30% Smoking~35% Unbalanced Diets Too Many Calories: Obesity Too Little Fiber & Micronutrients~20% Chronic Infections Mostly in Poor Countries~20% Hormones Breast, Endometrial, Etc.~2% Occupation<1% Pollution Mostly Heavy Air Pollution

The Causes of Cancer

Total = 107% because of multiple causes

Epithelial

(Block, Patterson and Subar, Nutr. Canc., 18: 1-29, 1992)

Fruits and Vegetables Protect Against Cancer

Relative Risk (Median)Cancer Site

Fraction of Studies Showing a Protective Effect (p=0.05)

LungOral LarynxEsophagusStomachPancreasCervixBladderColorectalMiscellaneous

BreastOvary/EndometriumProstate

Hormone-Dependent

24/259/94/4

15/1617/199/117/83/5

20/356/8

2.22.02.32.02.52.82.02.11.9-

8/143/4

4/14

1.31.81.3

Energy Sources 1999-2000Energy Sources 1999-2000 Cumulative Cumulative

FoodFood Percentage Percentage

1. SOFT DRINKS 7.1

2. CAKE, PASTRIES, & DOUGHNUTS 10.6

3. HAMBURGERS & CHEESEBURGERS 13.8

4. PIZZA 16.8

5. POTATO CHIPS, CORN CHIPS, POPCORN 19.7

6. RICE 22.4

7. ROLLS, BUNS, MUFFINS, & BAGELS 25.0

8. CHEESE & CHEESE SPREAD 27.6

9. BEER 30.2

10. FRENCH FRIES, FRIED POTATOES 32.4

Micronutrient Undernutrition in Americans

25%50%90; 75 mgMen; Women C

5; ~10-25%10-20; 25-50 %2.4 mcgMen; Women B12

25%; 50%75%400 mcgMen; Women Folate**

10% 50%1.7; 1.5 mgMen; Women B6

Vitamins

5-10% 25%8 mgWomen 50+ years

25% 75%18 mgWomen 20-30 years Iron

Minerals

<50% RDA

% ingesting

< RDA Population GroupNutrient

•Wakimoto and Block (2001) J Gerontol A Biol Sci Med Sci. Oct; 56 Spec No 2(2):65-80.

** Before U.S. Food Fortification

RDA % ingesting

Zinc Men; Women 50+ years 11; 8 mg 50% 10%

dUMP dTMP

Methionine

SHMT

B6

Serine

MTHFR

Homocysteine

(polymorphism)

TS

MS

CH2=THF

B12CH3-THF

Micronuclei in: RNA positive reticulocytes RNA negative erythrocytes

Folic AcidFolinic Acid

TIME (DAYS)1 yearpreRx

Normalrange

Mic

ronu

clei

per

100

0 ce

lls

20

50 100 150 200 250 300 350

0

30

40

80

130

Presupplementation Postsupplementation

1

10

100

Uracil/10,000 thymine

10

100

Micronuclei(mn reticulocytes/1000

reticulocytes)

Erythrocyte Folate (ng/ml)

10% US population50% Low income minority

<140 175-200 240-300 >300

Erythrocyte Folate (ng/ml)

<140 175-200 240-300 >300

xx

PLASMA FOLATE (NG/ML)

MIN

PC

Es/

1000

PC

Es

60

50

40

30

20

10

00 5 10 15 20 25 30

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10 12

Vitamin B-6 Intake

Human Lymphocyte DNA Strand Breaks (Comet Assay) vs. B-6 Intake

Folate, Vitamin B12, Homocysteine Status and Chromosome Damage Rate in Lymphocytes of Older Men

Michael F. Fenech, Ivor E.Dreostl and Josephine R., RinaldiCarcinogenesis Vol 13, pp.1329 - 1336, 1997

Folate, Vitamin B12, Homocysteine Status and DNA Damage in Young Australian AdultsMichael Fenech, Claire Aitken and Josephine RinaldiCarcinogenesis Vol 19, pp.1163 - 1173, 1998

Micronucleus Frequency in Human Lymphocytes is Related to Plasma Vitamin B12 and HomocysteineMichael F. FenechMutation Research 42:299-304, 1999

bna_ 0011_008.ppt

In a series of studies, we have been able to confirm that the micronucleus index in cytokinesis-blocked lymphocytes is significantly negatively correlated with plasma vitamin B12 (B12) concentration and significantly positively correlates with plasma homocysteine (HC). Furthermore we have shown in a randomized double-blind placebo-controlled dietary intervention study that intake of 3.5 times the RDI of folic acid and B12 significantly reduces the micronucleus index only in those with above average levels of micronucleus frequency. Micronucleus frequency is minimized when plasma HC is below 7.5 µmol/l and plasma B12 is above 300 pmol/l. Therefore, it is important to take account of the effect of B12 and HC when using the micronucleus assay for human biomonitoring studies. ©1999 Elsevier Science B.V. All rights reserved

CSIRO Human Nutrition, PO Box 100451 Gouger Street, Adelaide, SA 5000, Australia

Mean uracil content in sperm DNA from 23 men on diets low in fruits and

vegetables

14

12

10

8

6

4

2

0

DC uracil in DNA

Folate Deficiency Study epididymal Sperm Count

100

50

0

Epididymal Sperm

0.07

Seminal plasma folates vs. semen quality

Correlation coefficient (r); n=48

Total Sperm Count (105)Seminal Plasma Sperm Density (106/mL)

Non-methyl THF(methylene-THF, etc.)

5-Methyl THF

*p<0.05

* 0.31* 0.37

0.08

Vitamin B6 Deficiency

Mean intake by ethnic group (mg)20 yrs 40 yrs 60 yrs

RDA 1.3 1.3 1.7

White 2.3 2.2 2.1Black 2.4 1.9 1.5Hispanic 2.3 2.1 1.7

Mean intake by ethnic group (mg)20 yrs 40 yrs 60 yrs

RDA 1.3 1.3 1.5

White 1.5 1.4 1.7Black 1.5 1.2 1.3Hispanic 1.6 1.4 1.3

. 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.

Analysis of nonlinear regression models: comparison of an overall model and individual models of Z-transformed values vs. ln- nonheme liver iron

Over all

DCF-PMNs

DCF-Lymph

Rh123-PMNs

Rh123-Lymph

mtDNA damage

1/RCR

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

LN nonheme Fe (µmol/g wet liver)

Z s

co

re

normal

Heme deficiency induces oxidative stress

Succ-CoA + Gly ALA

2ALA

PBGOther intermediatesCytosol

PPIX PPGIX

protoheme

FeII

MitochondriaFC

Synthesis of Heme

9) Atamna et al (2001) JBC. 10) Atamna et al (2002) ABB. 11) Atamna et al (2002) PNAS.

Similarity Between the Consequences of Heme Deficiency and Normal Aging/neurodegeneration

Factor in Study Heme Deficiency Aging/Neurodegeneration

Complex IVIronOxidative StressAPP

NOSCell-cycle anddifferentiationMetabolismCalciumFerrochelataseHeme synthesis

Loss of complex IVAccumulationIncreasedDecreased andaggregate appearIncreasedDisabled differentiationor proliferationMitochondrial declineCorruptedIncreasedDecreased

Loss of complex IVAccumulationIncreaseddimmer or aggregate

IncreasedLoss of Axons; neuronal deathHypometabolismCorruptedIncreased in senescent cellsDecreased with age**

9

99

9,10

911

11

10

11

11

9*

*Not Determined in vivo. **Not determined in the aging brain

1994-96, 1998 CSFII Study, USDA

20

40

6080

100

120

140

Control ZnAD ZnDF

DC

F F

luo

resc

ence

In

ten

sity

(R

FU

)

Zinc Deficiency Induces IncreasedOxidative Stress in C6 Glioma Cells

*

Zinc Deficiency Induces Fapy Glycosylase (Fpg)-sensitive Single Strand Breaks in Human Lung Fibroblasts

0

40

80

120

160

200

Control ZnAD ZnDF

Co

met

Sco

re

Control (+Fpg) ZnAD (+Fpg) ZnDF (+Fpg)

*

Zinc Deficiency

Mean intake by ethnic group (mg)20 yrs 40 yrs 60 yrs

RDA 11 11 11

White 15 14 13Black 16 13 10Hispanic 15 15 11

Mean intake by ethnic group (mg)20 yrs 40 yrs 60 yrs

RDA 8 8 8

White 9 10 10Black 10 8 8Hispanic 11 9 8

“The main distinguishing characteristicbetween man and the lower animals

is the desire to take pills”

Mark Twain

1994-96, 1998 CSFII Study, USDA

ACKNOWLEDGEMENTS Children’s Hospital Oakland Research Institute

University of California at Berkeley

Dr. Hani AtamnaDr. Ronit Erlitski, Dr. David KillileaMs. Susan Mashiyama, Dr. Lynn Wallock,Dr. Patrick Walter Dr. Arnold Huang, Dr. Mitch KnutsonDr. Chantal CourtemancheDr. Emily Ho, Prof.Fernando Viteri

High-dose vitamin therapy stimulates variant enzymes with decreased

coenzyme-binding affinity (increased Km): Relevance to genetic

disease and polymorphisms

Am J Clin Nutr 2002; 75:616-658

Bruce N Ames, Ilan Elson-Schwab, and Eli A Silver,

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.

Summary of Work

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

11. Tetrahydrobiopterin12. S-Adenosyl Methionine13. Pantothenic Acid14. Lipoic Acid15. Carnitine16. Hormones17. Amino Acids18. Metals19. Maxi B Vitamins

1. Pyridoxine (Vitamin B6)2. Thiamine (Vitamin B1)3. Riboflavin (Vitamin B2)4. Niacin (Vitamin B3)5. Biotin (Vitamin B7)6. Cobalamin (Vitamin B12)7. Folic Acid8. Vitamin K9. Vitamin D10. 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

O2 O2-

e- e- e- e-

H2O2 •OH H2O

2

MitochondrialMatrix

Cellular Cytoplasm

Mitochondrial Outer MembraneMitochondrial Outer Membrane

Inner MembraneInner Membrane

IntermembraneSpace

VV

IIIIII

IIIIIIIVIV

CytCCytCCoQCoQ

HH++

PyruvateDehydrogenasecomplex

Citrate Synthase

-Ketoglutarate Dehydrogenase

Complex

Fumarate

L-Malate

Oxaloacetate

Acetyl-Co-A Citrate Isocitrate

-Ketoglutarate

Succinyl-Co-A

Succinate

FADFADH2

CITRICACID

CYCLE

H2OO2 ADP

ATP

ATP

NADH

NADH

NADH

NAD+

NADHNADH

HH++

HH++

HH++ HH++

HH++HH++HH++

HH++

HH++HH++

HH++ HH++HH++

HH++

HH++

HH++

HH++ HH++HH++

HH++

HH++

HH++

9

Estimated oxidative DNA adducts per rat liver cell

0

Old (26-mo)

Young (4-mo)

70,000

60,000

50,000

40,000

30,000

20,000

10,000 24,000

67,000

E. Stadtman, Science 257, 1220-1224 (1992)

carbonyl content(nmol/mg protein)

In carbonyl content(nmol/mg protein)

6

5

4

3

2

2

1

0

1000

Years

20 40 60 80

Months

3 12 20 26 *

Effect of ALCAR Supplementation on Cardiolipin Levels

Young

Ca

rdio

lipin

(µ

g p

er 1

0 c

ells

) 30

20

10

0

14

+ ALCAR

Old

R123 Fluorescence in Young and Old Rat Hepatocytes

YOUNG

OLD

WITHALCAR

WITHALCAR

NOALCAR

NOALCAR

No

rma

lize

d C

ell

Nu

mb

er

No

rma

lize

d C

ell

Nu

mb

er

Lipoic Acid Lowers Mitochondrial Oxidants in Old RatsF

l. U

nit

s/O

2 C

on

sum

ed p

er M

inu

te

20

10

0Young Old

17

+ LA+ LA

**

MDA levels in young and old rats with LA, ALCAR, or both

Young Old

MD

A (

pm

ol/m

g p

rote

in)

60

0

50

10

40

30

20

70

80

P<0.01P<0.05

***

+ L

A

+ A

LC

AR

+ A

LC

AR

+ L

A

+ L

A

+ A

LC

AR

+ A

LC

AR

+ L

A

20

***p<0.001 vs. young rat group

0

10

20

30

p<0.01

T c

ell

stim

ula

tio

n i

nd

ex

Young

Young Tre

ated

Old T

reat

edOld

Age-associated decrease in immune function and the effect of ALCAR (0.2%) + LA (0.1%) treatment for 2 months. Values are mean + SEM of 10-11 animals.

P<0.001

Ambulatory Activity before and After Supplementation with Lipoic Acid (LA) + Acetyl-L-Carnitine (ALCAR)

0

200

400

600

800

+ L

A +

AL

CA

R

OldYoung

+ L

A +

AL

CA

R

*

Dis

tan

ce

Tra

vel

ed

(c

m/h

ou

r/d

ay

)

*#

#

vs. young

vs. old

*

P<0.001

20

100

80

60

40

Spatial Memory Tested With Morris Water Maze

Young Old Old old Old 0

P<0.05

23

+ ALCAR + LA

+ ALCAR + LA

0.00

2.00

4.00

6

8

10

12

14

SOUND: Time to Signal

0 50 100 150 200

Young

Old

Old + ALCAROld + LA

Old + ALCAR + LA

0.00

2.00

4.00

6.00

8.00

10.00

12.00

LIGHT: Time to Signal

0 50 100 150 200

Peak procedure: for measuring temporal memory. Associated with striatum, cerebellum, & hippocampus

PEAK RATE: measures learning and motivation.

PEAK TIME: measures internal clock, food is rewarded only when animals push lever 40s after sound or light 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 RNA)

RNA is Oxidized

(92% is removed by RNase)

*oxo8G: 8-hydroxyguanosine; oxo8dG: 8-hydroxy-2’-deoxyguanosine27

Meta-analysis of acetyl-L-carnitine versus placebo for mild cognitive impairment and mild Alzheimer’s disease

Montgomery, S.A., Thal, L.J., and Amrein, R., Int. Clin. Psychopharmacol 18:61-71 (2003)

* #

nm

ol/m

in/g

1200

800

400

0

#

M

150

100

50

0Km for ALCAR

Km for CoA

**

##

#**

403020100-10

5

10

0

5

10

0200150100500-50

1/[ALCAR, mM] 1/[CoA, mM]

Vmax

young old = vs. young

# = vs. old

1/v 1/v

*

B

CC

A

29

80

75

70

65

60

55

50

45

401900 1910 1920 1930 1940 1950 1960 1970 1980 1990

46.4

4950.1

53.654.5

56.3

5861.3 61.4

65.7 65.666.7

71.173.2

74.9

77.578.9

71.469.9

67.1

Life Expectancy of Men and Women at Birth

SOURCE: National Institute on Aging

ACKNOWLEDGEMENTSChildren’s Hospital Oakland Research Institute

University of California at BerkeleyUniversity of California at Irvine

Linus Pauling Institute, Oregon State University

Dr. Jiankang LiuDr. Tory HagenDr. Afshin GharibDr. David KillileaDr. Patrick WalterDr. Hani AtamnaDr. Emily Ho

Dr. Elizabeth HeadDr. Carl W. Cotman

35