Bruce N. AmesChildren’s Hospital Oakland Research InstituteProfessor, University of California, Berkeley

Delaying (or Accelerating) the Mitochondrial Decay of Aging

Timiras ‘06JA25

O2 O2-

e- e- e- e-

H2O2 •OH H2O

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

Base Excision Repair:Specific DNA Glycosylase removes base.

Nucleotide Excision Repair: Exonuclease removes

stretch of DNA

DAMAGED BASE

DAMAGED DEOXYNUCLEOSIDE

Base is Damaged

TOURINE

3

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

10,00024,000

67,000

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

carbonyl content(nmol/mg protein)

carbonyl content(nmol/mg protein)

6

5

4

3

2

2

1

0

1000

Years

20 40 60 80

Months

3 12 20 26 *

MDA (pmol/mg

protein)

Young

Old

160

140

120

100

80

60

40

20

0

Brain Liver Heart Kidney Lung

* *

*

**

6

Proc. Natl. Acad. Sci. USAVol. 91, pp. 10771-10778, November 1994

Review

Oxidative damage and mitochondrial decay in aging (bioenergetics / mitochondrial DNA / cardiolipin / acetyl-L-carnitine / neurodegeneration)

Mark K. Shigenaga, Tory M. Hagen, and Bruce N. Ames*Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California, Berkeley, CA 94720

Contributed by Bruce N. Ames, July 27, 1994

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

Young

Old

Mitochondria in hippocampal neurons

Electron Microscopy Images

Mitochondria from old rats compared to those from young rats:

1) Lower Cardiolipin

2) Lower Membrane Potential

3) Lower Oxygen Utilization

4) Increased Oxidant Leakage

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

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

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 Rats

Fl.

Un

its

/O2 C

on

sum

ed p

er M

inu

te

20

10

0Young Old

+ 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

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

*

0

10

20

30

p <0.01

T c

ell

sti

mu

lati

on

ind

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

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

+ 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

* #

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

*

Young

Old

YC YT OC OT0

10

20

30

40

50

60

70

80 #*

Young Old OldALCAR+LA

YoungALCAR+LA

Age-related loss of undamaged mitochondria

in hippocampal neurons

Electron Microscopy Images

New Yorker, June 6, 2005

“You’re fifty-seven years old. I’d like to get that down a bit.”

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)

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

symptomatic diabetic neuropathy

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

61

79

60

72

6666

60

25

48

55

0

10

20

30

40

50

60

70

80

90

ALADIN I ALADIN III SYDNEY NATHAN II Meta-Analysis

Pe

rcen

t

-lipoic acidplacebo

* p<0.05 vs Placebo

ITT analysis of 4 phase II-III RCTs plus meta-analysis: 600 mg I.v. per day for 3 weeksTotal Symptom Score (TSS): relative improvement at 3 weeks vs baseline

n= 77 81 338 165 60 60 241 236 716 542

Micronutrient Undernutrition in Americans

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

** Before U.S. Food Fortification

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 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

An overview of evidence for a causal relationship between iron deficiencyduring development and cognitive or behavioral function in children

Joyce C McCann and Bruce N Ames(2006) Submitted

Is docosahexaenoic acid, an n3 long-chain polyunsaturated fatty acid, required for development of normal brain function? An overview of evidence from cognitive and behavioral tests in humans and animals

Joyce C McCann and Bruce N AmesAmerican Journal of Clinical Nutrition (2005) 82:281-95

An overview of evidence for a causal relationship between dietary availability of choline during development and cognitive function in offspring

Joyce C McCann, Mark Hudes, and Bruce N AmesNeuroscience & Biobehavioral Reviews, (2006) in press

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)

*

Succ-CoA + Gly ALA

2ALA

PBG Porphyrins

Cytosol

PPIX PPGIX

Heme

FeII

MitochondriaFC

Synthesis of Heme

Heme-a

PLP

ALA

CH

CH2

CH2

CH2

COOH COOH

CH2

CH2

CH3

CH3

H3C

Fe

N N

N N

CH CH

CH

CH

H3C

CH

Protoheme

CH2

(1) Farnesylation

(2) Oxidation

Heme-bHeme-a

(Only in complex IV)

Maturation of heme-b to heme-a is rate limiting for the assembly of complex IV

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

Effect of Biotin on Cell Senescence

0.0 2.5 5.0 7.5 10.0 12.5 15.020253035404550556065

cdFBS-BD

nFBS-BSnFBS-+B--BS

Weeks

cdFBS+B-BS

Biotin deficiency accelerates cell senescence

Micronutrient

Heme-a

Complex IV

Oxidative Stress

DNA Damage

Early Agin

g

Pyridoxine [++]

Zinc +? ++ ++ ++

Riboflavin

Iron ++ ++ ++ ++

Copper ++ [++]

Biotin ++ ++ ++ ++

Lipoic Acid [++]Pantothenat

e [++]

Micronutrient requirements for heme synthesis

++ = Atamna/Ames [++] Literature

Magnesium Deficiency Shortens Fibroblast Lifespan

Magnesium Deficiency Induces Senescence Marker

TRIAGE HYPOTHESIS

• In human cells in culture, the micronutrientdeficiencies tested cause DNA damage and earliersenescence.

• Throughout evolution animals have been facedwith shortages of individual micronutrients of the~ 40 that are required. On deficiency evolutionwould favor survival over long term health.

• We hypothesize that metabolism required for longterm health, e.g. DNA protective systems, is lost ondeficiency before metabolism required for theshort term, e.g. making ATP.

• If our hypothesis is true for humans, ensuringmicronutrient adequacy may be important forincreasing longevity and minimizing defects indevelopment, DNA damage, and early aging.

The Economist, December 13, 2003

Energy Sources - 1999-2000Food

1. Regular soft drinks 7.1

2. Cake, sweet rolls, doughnuts, pastries 10.6

3. Hamburgers, cheeseburgers, meatloaf 13.8

4. Pizza 16.8

5. Potato chips, corn chips, popcorn 19.7

6. Rice 22.4

7. Rolls, buns, English muffins, bagels 25.0

8. Cheese or cheese spread 27.6

9. Beer 30.2

10. French fries, fried potatoes 32.4

Gladys Block from National Health and Nutrition Examination Survey (NHANES) 2000.

Cumulative Percentage

CAUTION: HAZARDOUS WAIST

Visceral fat increases your risk of heart disease, diabetes, cancer, etc.

Start a waist disposal program today!

Dr. Allen Spiegel, NIDDK/NIH

“The main distinguishing characteristicbetween man and the lower animals

is the desire to take pills”

Mark Twain

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

END

dUMP dTMP

Methionine

SHMT

B6

Serine

MTHFR

Homocysteine

(polymorphism)

TS

MS

CH2=THF

B12CH3-THF

Micronuclei in: RNA positive erythrocytes RNA negative erythrocytes

Folic AcidFolinic Acid

TIME (DAYS)

1 yearpreRx

Normalrange

Mic

ron

ucl

ei p

er 1

000

cell

s

20

50 100 150 200 250 300 350

0

30

40

80

130

xx

PLASMA FOLATE (NG/ML)

MIN

PC

Es/

1000

PC

Es 60

50

40

30

20

10

00 5 10 15 20 25 30

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

Michael Fenech, Ivor Dreostl, and Josephine Rinaldi, Carcinogenesis 13:1329-1336, 1997

Folate, Vitamin B12, Homocysteine Status and DNA Damage in Young Australian Adults

Michael Fenech, Claire Aitken, and Josephine Rinaldi, Carcinogenesis 19:1163 - 1173, 1998

Micronucleus Frequency in Human Lymphocytes is Related to Plasma Vitamin B12 and Homocysteine

Michael Fenech, Mutation Research 42: 299 - 304, 1999

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 correlated 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.