Effect of dietary administration of lipoic acid on protein oxidation in rat liver

1
Cumulative Net Risk of Death from Cancer for Rat
and Human
30
Rat
Human
20
Cumulative Risk ()
10
0
0
1
2
20
40
60
80
100
AGE (years)
2
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3
2
4
Estimated oxidative DNA adducts per rat liver cell
Old (26-mo)
70,000
60,000
67,000
50,000
40,000
30,000
Young (4-mo)
20,000
24,000
10,000
0
5
E. Stadtman, Science 257, 1220-1224 (1992)
6
Cellular Cytoplasm
Mitochondrial Outer Membrane
Intermembrane Space
I
CoQ
Inner Membrane
CytC
III
II
V
IV
NADH
Mitochondrial Matrix
NAD
FAD
O2
FADH2
ADP
H2O
Succinate
ATP
Fumarate
ATP
Succinyl-Co-A
?-Ketoglutarate Dehydrogenase Complex
NADH
L-Malate
NADH
?-Ketoglutarate
Pyruvate Dehydrogenase complex
NADH
Oxaloacetate
NADH
Isocitrate
Citrate
Acetyl-Co-A
Citrate Synthase
9
7
Intermembrane Space
Matrix
Carnitine acyltransferase II
Carnitine
Carnitine
CoA-SH
CoA-SH
Carnitine acyltransferase I
12
8
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
9
Effect of ALCAR Supplementation on Cardiolipin
Levels
ALCAR
Old
Young
14
10
R123 Fluorescence in Young and Old Rat
Hepatocytes
YOUNG
NO ALCAR
Normalized Cell Number
WITH ALCAR
OLD
NO ALCAR
WITH ALCAR
Normalized Cell Number
11
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
12
Lipoic Acid Lowers Mitochondrial Oxidants in Old
Rats

20
Fl. Units/O2 Consumed per Minute
10
LA
LA
0
Young
Old
17
13
R-Lipoic Acid Restores Cellular Ascorbate
8
6
Ascorbate (nmol/105 cells)
4
ALCAR LA
ALCAR LA
ALCAR
ALCAR
0
Old
Young
18
14
Effect of R-Lipoic Acid LA Supplementation on
GSH Levels ALCAR
20
10
GSH (nmol/106 cells)
LA ALCAR
LA ALCAR
LA
LA
0
Young
Old
19
15
MDA levels in young and old rats with LA, ALCAR,
or both
80
Plt0.05
plt0.001 vs. young rat group
Plt0.01
70
60

Young
Old
MDA (pmol/mg protein)
50
40
30
20
10
0
LA
LA
ALCAR
ALCAR LA
ALCAR
ALCAR LA
20
16
10
10
1/v
1/v
5
5
0
0
40
30
20
10
0
-10
200
150
100
50
0
-50
1/ALCAR, mM
1/CoA, mM
young
old
vs. young

vs. old



Vmax
17
A
B
C
C
29
18
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.
Plt0.001
3
0
p
lt
0
.
0
1
2
0
T cell stimulation index
1
0
0
Old
Young
Old Treated
Young Treated
19
Ambulatory Activity before and After
Supplementation with Lipoic Acid (LA)
Acetyl-L-Carnitine (ALCAR)
vs. young

vs. old


Distance Travelled (cm/hour/day)

20
Morris Water Maze for Testing Spatial Memory
Spatial Memory relies on intact hippocampal
function. Treatments improved poor memory in old
rats
22
21
Spatial Memory Tested With Morris Water Maze
100
Plt0.001
Plt0.05
80
60
40
LA
ALCAR
ALCAR LA
20
0
Young
Old
Old
old
Old
23
22
Improvements in Spatial Memory of Old Rats
Treated with ALCAR, LA, or Both

Time (sec)
Old ALCAR LA
Old ALCAR
Old LA
Young
Old
Old ALCAR LA
Old ALCAR
Old LA




Young
Old
24
23
Peak procedure for measuring temporal memory.
Associated with striatum, cerebellum,
hippocampus
14
PEAK RATE measures learning and motivation.
12.00
Young
12
Old
10.00
Old ALCAR
PEAK TIME measures internal clock, food is
rewarded only when animals push lever 40s after
sound or light signal
Old LA
10
Old ALCAR LA
8.00
8
6.00
6
4.00
4.00
2.00
2.00
0.00
0.00
SOUND Time to Signal
LIGHT Time to Signal
25
24
Oxidative Damage to Nucleic Acid in Old Rats by
mAb to oxo8G/oxo8dG Immunohistochemical stain of
neurons
26
25
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-deoxyguanosine
27
26
Dendritic spine density of cortical neurons in
rat brain with Golgi staining Effects of 11
month treatment with ALCAR, LA or ALCARLA
70
60
50
Spine density
40
30
20
10
0
Young
Old
ALCAR
LA
ALCAR LA
Liu, Mervis, and Ames, Unpublished data
27
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 1861-71 (2003)
28
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)
29
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30
Life Expectancy of Men and Women at Birth
80
77.5
78.9
74.9
75
73.2
71.1
71.4
69.9
70
66.7
67.1
65.7
65.6
65
61.4
61.3
58
60
56.3
54.5
55
53.6
50.1
49
50
46.4
45
SOURCE National Institute on Aging
40
1900
1910
1920
1930
1940
1950
1960
1970
1980
1990
31
ACKNOWLEDGEMENTS
Childrens 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 Dr. Afshin
Gharib Dr. David Killilea Dr. Patrick Walter Dr.
Hani Atamna Dr. Emily Ho
Dr. Elizabeth Head Dr. Carl W. Cotman
35
32
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33
Micronutrient Undernutrition in Americans
lt50 RDA
ingesting lt RDA
Population Group
Nutrient
ingesting
RDA
Minerals
25
75
18 mg
Women 20-30 years
Iron
5-10
25
8 mg
Women 50 years

Men Women 50 years
11 8 mg
50
10
Zinc
Vitamins
10
50
1.7 1.5 mg
Men Women
B6
25 50
75
400 mcg
Men Women
Folate
5 10-25
10-20 25-50
2.4 mcg
Men Women
B12
25
50
90 75 mg
Men Women
C
Wakimoto and Block (2001) J Gerontol A Biol Sci
Med Sci. Oct 56 Spec No 2(2)65-80. Before
U.S. Food Fortification
34
Analysis of nonlinear regression models
comparison of an overall model and individual
models of Z-transformed values vs. ln- nonheme
liver iron
. 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.
35
Synthesis of Heme
Cytosol
PBG
Other intermediates
2ALA
PPIX
PPGIX
FeII
FC
protoheme
Mitochondria
Succ-CoA Gly ALA
36
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37
Heme deficiency induces oxidative stress
38
Similarity Between the Consequences of Heme
Deficiency and Normal Aging/neurodegeneration
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.
39
ADJUSTED ODDS RATIO FOR INADEQUATE
PREGNANCY OUTCOME AMONG ANEMIC PREGNANT WOMEN
Source Scholl et al., AJCN 1992

3


2

ODDS RATIO
1
0
SMALL GEST. AGE
LBW
PRE TERM
POOR WEIGHT GAIN
40
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41
ACUTE/SEVERE DEFICIENCY
OVERLOAD
42
Iron Deficiency
43
(No Transcript)
44
Zinc Deficiency Induces IncreasedOxidative
Stress in C6 Glioma Cells

140
120
100
Intensity (RFU)
DCF Fluorescence
80
60
40
20
Control
ZnAD
ZnDF
45
Zinc Deficiency Induces Fapy Glycosylase
(Fpg)-sensitive Single Strand Breaks in Human
Lung Fibroblasts
Control (Fpg)
ZnAD (Fpg)
ZnDF (Fpg)

200
160
120
Comet Score
80
40
0
Control
ZnAD
ZnDF
46
Zinc Deficiency
47
Magnesium Deficiency
48
Human Lymphocyte DNA Strand Breaks (Comet Assay)
vs. B-6 Intake
3
.
5
3
.
0
2
.
5
2
.
0
Avg 100 comets
1
.
5
1
.
0
0
.
5
0
.
0
B
a
s
e
l
i
n
e
0
.
7
1
.
4
2
.
2
1
0
.
3
Vitamin B-6 Intake (mg/d)
T. Shultz, C. Hansen, K. Hunt, J. Leklem, A.
Huang, B. Ames
49
Human Lymphocyte DNA Strand Breaks (Comet Assay)
vs. B-6 Intake
3.5
3
2.5
2
1.5
1
0.5
0
0
2
4
6
8
10
12
Vitamin B-6 Intake
50
The main distinguishing characteristic between
man and the lower animals is the desire to take
pills Mark Twain
51
ACKNOWLEDGEMENTS
Childrens Hospital Oakland Research
Institute University of California at Berkeley

Dr. Hani Atamna Dr. Ronit Erlitski, Dr. David
Killilea Ms. Susan Mashiyama, Dr. Lynn
Wallock, Dr. Patrick Walter Dr. Arnold Huang,
Dr. Mitch Knutson Dr. Chantal Courtemanche Dr.
Emily Ho, Prof.Fernando Viteri