cumulative net risk of death from cancer for rat and human cumulative risk (%) 30 20 10 0 rat human...

Cumulative Net Risk of Death from Cancer for Rat and HumanC

um

ula

tive

Ris

k (%

)30

20

10

0

Rat Human

AGE (years)

0 1 2 20 40 60 80 100

O2 O2-

e- e- e- e-

H2O2 •OH H2O

2

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 *

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

Carnitine acyltransferase II

MatrixIntermembrane Space

Carnitine acyltransferase I

Carnitine

Carnitine

R C

O

S-CoA

Carnitine

R C

O

CoA-SH

R C

O

S-CoA

Carnitine

R C

O

CoA-SH

12

L-Carnitine/Acetyl-L-Carnitine (ALCAR)

• Mediates the ratio of acetyl-CoA/CoA

• Decreases with age in plasma and in brain

• Improves cognitive function in rats12

• Transports long-chain fatty acids into mitochondria

• Removes short- and medium-chain fatty acids that accumulate

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

R--Lipoic Acid (LA) in mitochondria

• LA reduced to dihydrolipoic acid, a potent antioxidant, & chelator of Fe & Cu• Coenzyme of pyruvate and -ketoglutarate dehydrogenases• Involved with carbohydrate utilization for ATP production• Improves cognitive function in aged mice

15

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

**

R-Lipoic Acid Restores Cellular Ascorbate

As

co

rba

te (

nm

ol/1

05 c

ells

)

6

0Young Old

4

8

AL

CA

R +

LA

AL

CA

R

AL

CA

R

AL

CA

R +

LA

18

Effect of R-Lipoic Acid [LA] Supplementation on GSH Levels + ALCAR

GS

H

(n

mo

l/10

6 c

ells

)

10

0Young Old

20

+ L

A +

AL

CA

R

+ L

A

+ L

A

+ L

A +

AL

CA

R

19

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

* #

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

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%) treatement 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

led

(c

m/h

ou

r/d

ay) *

#

#

vs. young

vs. old

*

Spatial Memory relies on intact hippocampal function.

Treatments improved poor memory in old rats

Morris Water Maze for Testing Spatial Memory

22

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

Improvements in Spatial Memory of Old Rats Treated with ALCAR, LA, or Both

0

5

10

15

20

25

Platform quadrant Platform position

Tim

e (s

ec) **

*

***

# # ##

You

ng

Old

Old

+ A

LC

AR

Old

+ L

AO

ld +

ALC

AR

+ L

A

You

ng

Old

Old

+ A

LC

AR

Old

+ L

AO

ld +

ALC

AR

+ L

A

24

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

Dendritic spine density of cortical neurons in rat brain with Golgi staining: Effects of 11 month treatment with ALCAR, LA or ALCAR+LA

Young Old +ALCAR +LA +ALCAR+LA

0

10

20

30

40

50

60

70

***

Liu, Mervis, and Ames, Unpublished data

Sp

ine

den

sity

Meta-analysis of acetyl-L-carnitine versus placebo for mild cognitive impairment and mild Alzheimers disease

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

Treatment with alpha-lipoic acid significantly improves both neuropathic symptoms and deficits in diabetic patients with

symptomatic diabetic neuropathy

Professor Daniel Ziegler of the Diabetes Research Institute, Düsseldorf, Germany: Meta-Analysis Provides Highest Level of Evidence, Diabetes Monitor (2002, p6)

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

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%

. 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

Succ-CoA + Gly ALA

2ALA

PBGOther intermediatesCytosol

PPIX PPGIX

protoheme

FeII

MitochondriaFC

Synthesis of Heme

Loss of activity of complex IV in heme-deficient cells

Heme deficiency induces oxidative stress

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

0

1

2

3

LBW PRETERM

SMALLGEST. AGE

POORWEIGHT

GAIN

*

**

*

Anemic Fe Def Anemic Other Anemic

ADJUSTED ODDS RATIO FOR INADEQUATE PREGNANCYOUTCOME AMONG ANEMIC PREGNANT WOMEN

Source: Scholl et al., AJCN 1992

OD

DS

RA

TIO

: significant*

ACUTE/SEVEREDEFICIENCY

OVERLOAD

Iron Deficiency

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

RDA 8 8 8

White 18 19 17Black 18 16 13Hispanic 17 16 15


RDA 18 18 8


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


RDA 11 11 11



RDA 8 8 8


Magnesium Deficiency


RDA 400 420 420



RDA 310 320 320


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Baseline 0.7 1.4 2.2 10.3

Av

g 1

00

co

me

ts ZE-1TK-2AW-4TB-5MR-6LS-7AB-8CH-9RB-12YHC-13HP-16TS-10

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

Vitamin B-6 Intake (mg/d)

T. Shultz, C. Hansen, K. Hunt, J. Leklem, A. Huang, & B. Ames

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

“The main distinguishing characteristicbetween man and the lower animals

is the desire to take pills”

Mark Twain

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

cumulative net risk of death from cancer for rat and human cumulative risk (%) 30 20 10 0 rat human...

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