Transcript Effect of dietary administration of lipoic acid on protein oxidation in

Delaying (or Accelerating) the
Degenerative Diseases of Aging
O2
e-
O2-
e-
H2O2
e-
•OH
Bruce N. Ames
Children’s Hospital Oakland Research Institute
Professor, University of California, Berkeley
e-
H 2O
24JN07
Timiras
Base is
Damaged
Nucleotide Excision Repair:
Exonuclease removes
stretch of DNA
Base Excision Repair:
Specific DNA Glycosylase
removes base.
DAMAGED
DEOXYNUCLEOSIDE
DAMAGED
BASE
TO
URINE
3
Estimated oxidative DNA adducts
per rat liver cell
70,000
Old (26-mo)
60,000
67,000
50,000
40,000
30,000
20,000
10,000
0
Young (4-mo)
24,000
carbonyl content
(nmol/mg protein)
carbonyl content
(nmol/mg protein)
6
2
5
4
1
3
0
2
0
20
40
60
Years
80
100
3
12
20
26 *
Months
Source: E. Stadtman, Science 257, 1220-1224 (1992)
160
MDA
(pmol/mg
protein) 140
Young
*
Old
*
120
100
80
60
*
40
20
*
*
0
Brain
Liver
Heart
Kidney
Lung
6
Proc. Natl. Acad. Sci. USA
Vol. 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
Mitochondria in hippocampal neurons
Young
Old
Electron Microscopy Images
H+
Cellular Cytoplasm
H+
H+
H+
H+
H+
Mitochondrial Outer Membrane
H+
Intermembrane
Space
Mitochondrial
Matrix
H+
H+
CoQ
I
Inner Membrane
H+
NAD+
H+
H+
FAD
CytC
H+
Succinate
Fumarate
L-Malate
NADH
Pyruvate
NADH
Oxaloacetate
Dehydrogenase
complex
Acetyl-Co-A
Citrate
Synthase
ATP
CITRIC
ACID
CYCLE
Citrate
H+
H+
V
IV
H+
FADH2
H+
H+
III
II
NADH
H+
O2
H2O
H+
ADP
+
ATP H
Succinyl-Co-A
NADH
-Ketoglutarate
Dehydrogenase
Complex
-Ketoglutarate
Isocitrate
NADH
H+
H+
9
Cardiolipin (µg per 106 Cells)
Cardiolipin Levels in 3 and 24 Month Old Rat Hepatocytes
30
20
**
10
0
Young
Old
10
R123 Fluorescence in old and young rat hepatocytes
0.03
Normal cell number
Old
0.02
Young
0.01
0.00
10
100
1000
Fluorescence/cell
11
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)
• Transports long-chain fatty acids into mitochondria
• Removes short- and medium-chain fatty acids that accumulate
• Mediates the ratio of acetyl-CoA/CoA
• Decreases with age in plasma and in brain
• Improves cognitive function in rats
12
Cardiolipin (µg per 10 cells)
Effect of ALCAR Supplementation on Cardiolipin Levels
30
20
+ ALCAR
**
10
0
Young
Old
14
Normalized Cell Number
Normalized Cell Number
NO
ALCAR
YOUNG
WITH
ALCAR
OLD
NO
ALCAR
WITH
ALCAR
R123 Fluorescence
in Young and Old
Rat Hepatocytes
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
15
Fl. Units/O2 Consumed per Minute
Lipoic Acid Lowers Mitochondrial Oxidants in Old Rats
**
20
10
+ LA
+ LA
0
Young
Old
80
70
***p<0.001 vs. young rat group
P<0.05
P<0.01
60
50
Old ***
Young
40
30
20
+ ALCAR + LA
+ ALCAR
+ LA
+ ALCAR + LA
0
+ ALCAR
10
+ LA
MDA (pmol/mg protein)
MDA levels in young and old rats with LA, ALCAR, or both
20
800
*
*
vs. young
# vs. old
600
+ LA + ALCAR
400
#
+ LA + ALCAR
Distance Traveled (cm/hour/day)
Ambulatory Activity before and After Supplementation
with Lipoic Acid (LA) + Acetyl-L-Carnitine (ALCAR)
*
200
0
Young
Old
T cell stimulation index
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.
30
20
10
0
P <0.001
p <0. 01
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
+ ALCAR
+ LA
+ ALCAR
+ LA
20
0
Young
Old
Old
Old
Old
Peak procedure: for measuring temporal memory.
Associated with striatum, cerebellum, & hippocampus
14
12.00
PEAK RATE:
measures learning
and motivation.
12
10.00
PEAK TIME: measures
internal clock, food is
rewarded only when
animals push lever 40s
after sound or light signal
10
8
Young
Old
Old + ALCAR
Old + LA
Old + ALCAR + LA
8.00
6.00
6
4.00
4.00
2.00
2.00
0.00
0.00
0
50
100
150
SOUND: Time to Signal
200
0
50
100
150
LIGHT: Time to Signal
200
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
The Journal of Biological Chemistry
Vol. 278, pp. 8135-8145, March 7, 2003
Modulation of Gene Expression by Cancer Chemopreventive
Dithiolethiones through the Keap1-Nrf2 Pathway
IDENTIFICATION OF NOVEL GENE CLUSTERS FOR CELL SURVIVAL
Mi-Kyoung Kwak, Nobunao Wakabayashi, Ken Itoh, Hozumi Motohashi,
Masayuki Yamamoto, and Thomas W. Kensler
Proc. Natl. Acad. Sci. USA
Vol. 101, pp. 3381-3386, March 9, 2004
Decline in transcriptional activity of Nrf2 causes age-related loss
of glutathione synthesis, which is reversible with lipoic acid
Jung H. Suh, Swapna V. Shenvi, Brian M. Dixon, Honglei Liu, Anil K. Jaiswal,
Rui-Ming Liu, and Tory M. Hagen
Induction of Phase 2 Enzymes
250
Induced
231
Number of Genes
200
150
100
106
50
33
0
31
-50
30
Repressed
6h
24h
7d (diet)
Modulation of Nrf-2-dependent gene expression by D3T in mouse liver.
Kwak, et al. J bio Chem, 2003
10
10
1/v
1/v
5
5
0
0
-10
0
10
20
30
40
-50
0
1/[ALCAR, mM]
young
100
**
old
800
*
200
* = vs. young
150
#
150
1/[CoA, mM]
#
#
mM
nmol/min/g
1200
50
# = vs. old
#
100
**
400
50
#
0
0
Vmax
Km for
ALCAR
Km for
CoA
Age-related loss of undamaged mitochondria
in hippocampal neurons
Young
Old
Young
Electron Microscopy Images
Young
Old
Old
ALCAR+LA
ALCAR+LA
“You’re fifty-seven years old.
I’d like to get that down a bit.”
New Yorker, June 6, 2005
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
90
ITT analysis of 4 phase II-III RCTs plus meta-analysis: 600 mg I.v. per day for 3 weeks
Total Symptom Score (TSS): relative improvement at 3 weeks vs baseline
80
-lipoic acid
placebo
79
70
72
66
Percent
60
66
60
61
60
55
50
48
40
30
25
20
10
n= 77
81
338 165
60
60
241 236
716 542
0
ALADIN I
* p<0.05 vs Placebo
ALADIN III
SYDNEY
NATHAN II
Meta-Analysis
Source: Professor Daniel Ziegler of the Diabetes Research Institute, Düsseldorf, Germany:
Meta-Analysis Provides Highest Level of Evidence, Diabetes Monitor (2002, p6)
Micronutrient Undernutrition in Americans
Nutrient
Population Group
% Ingesting
< EAR *
From Food
Minerals
Iron
Women 14 - 50 years
16 %
Magnesium
All
56 %
Zinc
All
12 %
B6
Women > 70 years
49 %
Folate
Adult Women
16 %
E
All
93 %
C
All
31 %
Vitamins
* USDA What we Eat in America (NHANES 2001-2002) Sept. 2005
Methionine
Serine
B6
MTHFR
(polymorphism)
SHMT
B12
CH3-THF
MS
CH2=THF
TS
dUMP
Homocysteine
dTMP
Micronuclei in: RNA positive erythrocytes
RNA negative erythrocytes
Micronuclei per 1000 cells
130
Folic Acid
Folinic Acid
80
40
30
20
Normal
range
0
1 year
preRx
50
100
150
TIME (DAYS)
200
250
300
350
MIN PCEs/1000 PCEs
60
50
40
30
20
x
10
x
0
0
5
10
15
20
PLASMA FOLATE (NG/ML)
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.
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
Z score
1.5
DCF-Lymph
Rh123-PMNs
1
Rh123-Lymph
0.5
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.
An overview of evidence for a causal relationship
between iron deficiency
during development and cognitive or behavioral
function in children
Joyce C McCann and Bruce N Ames
(2006) in press
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 Ames
American 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 Ames
Neuroscience & Biobehavioral Reviews, (2006) 30:696-712.
Zinc Deficiency Induces Increased
Oxidative Stress in C6 Glioma Cells
DCF Fluorescence
Intensity (RFU)
*
140
120
100
80
60
40
20
Control
ZnAD
ZnDF
Zinc Deficiency Induces Fapy Glycosylase (Fpg)-sensitive
Single Strand Breaks in Human Lung Fibroblasts
Comet Score
Control (+Fpg)
ZnAD (+Fpg)
ZnDF (+Fpg)
*
200
160
120
80
40
0
Control
ZnAD
ZnDF
Synthesis of Heme
Cytosol
PBG
Porphyrins
Heme-a
2ALA
ALA
PPIX
FeII
PPGIX
FC
Mitochondria
Heme
Succ-CoA + Gly
PLP
ALA
Maturation of heme-b to heme-a is rate limiting for the
assembly of complex IV
otoheme
CH2
CH
H3C
N
CH
N
H3C
CH
N
CH3
CH
CH2
CH2
CH2
CH2
COOH
COOH
Heme-b
CH2
N
Fe
CH
(2) Oxidation
CH3
CH
(1) Farnesylation
Heme-a
(Only in complex IV)
H+
Cellular Cytoplasm
H+
H+
H+
H+
H+
Mitochondrial Outer Membrane
H+
Intermembrane
Space
Mitochondrial
Matrix
H+
H+
CoQ
I
Inner Membrane
H+
NAD+
H+
H+
FAD
CytC
H+
Succinate
Fumarate
L-Malate
NADH
Pyruvate
NADH
Oxaloacetate
Dehydrogenase
complex
Acetyl-Co-A
Citrate
Synthase
ATP
CITRIC
ACID
CYCLE
Citrate
H+
H+
V
IV
H+
FADH2
H+
H+
III
II
NADH
H+
O2
H2O
H+
ADP
+
ATP H
Succinyl-Co-A
NADH
-Ketoglutarate
Dehydrogenase
Complex
-Ketoglutarate
Isocitrate
NADH
H+
H+
9
Biotin deficiency accelerates cell senescence
Micronutrient deficiency and heme synthesis in human cell culture
Micronutrient
Deficiency
Heme
Deficit
Pyridoxine
[+]
Zinc
Complex IV
Deficit
Oxidative
Stress
DNA
Early
Damage Senescence
++
+
#
#
[+]
++
Riboflavin
Iron
+
+
[+]
Copper
[+]
+
[+]
[+]
+
+
[+]
[+]
[+]
Biotin
Lipoic Acid
Pantothenate
+
+ = Atamna/Ames, ++Askree /Ames, #Ho/Ames [+] Literature
+
Magnesium Deficiency Shortens Fibroblast Lifespan
Magnesium Deficiency Induces DNA-Protein Crosslinks
Calcium Deficiency
Fenech: chromosome breaks
Lipkin: colon cancer mice
Folate Deficiency
MacGregor/Ames/Fenech: chromosome
breaks mice/humans
Willett: epi colon cancer humans
Vitamin D Deficiency
Garland: epi colorectal cancer humans
Vitamin B12
Fenech: Chromosome breaks
Selenium
Rao: DNA damage
Combs/Trumbo: Cancer humans
Omega-3 FA
Denkins: Cancer
Magnesium Deficiency
Niacin
Bell: chromosome breaks humans
Larsson: epi colorectal cancer humans
Kirkland/Depeint: DNA damage
Zinc Deficiency
Choline
Fong: esophageal cancer humans/rodents
da Costa: DNA damage in humans
Potassium Deficiency
Chang: Cardiovascular Disease
Micronutrient deficiencies are found to cause DNA damage in mice
or human cells in culture and, where assayed, earlier senescence.
I hypothesize that DNA damage and late onset disease are a
consequence of a triage allocation response to micronutrient
shortage.
1) Episodic shortage of micronutrients were common throughout
evolution.
2) natural selection favors short-term survival at the expense of
long-term health when they are in conflict
3) this was achieved by allocating scarce micronutrients by
triage in part through an adjustment of the binding affinity of
each enzyme for its required micronutrient.
The consequences of the triage are evident at all levels. For
example, in metabolic reactions, ATP synthesis would be favored
over DNA-protecting enzymes; in cells, erythrocytes over
leukocytes; and in organs, the heart over the liver.
If this hypothesis is validated, ensuring micronutrient adequacy
in humans throughout life is essential for maximizing longevity and
The Economist, December 13, 2003
Energy Sources - 1999-2000
Food
Cumulative Percentage
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.
Dr. Allen Spiegel, NIDDK/NIH
CAUTION: HAZARDOUS WAIST
Visceral fat increases your risk of
heart disease, diabetes, etc.
Start a waist disposal program today.
“The main distinguishing characteristic
between man and the lower animals
is the desire to take pills”
Mark Twain
Life Expectancy of Men and Women at Birth
80
77.5
78.9
74.9
75
71.1
73.2
71.4
69.9
70
66.7
67.1
65.7 65.6
65
61.3 61.4
58
60
56.3
54.5
55
49
50
53.6
50.1
46.4
45
40
1900
1910
1920
1930
1940
1950
1960
1970
1980
1990
SOURCE: National Institute on Aging
END