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Changes in Acute Glutathione Levels in Human Plasma Following Lipoic Acid Supplementation
Shawn Johnson
Dr. Tory Hagen
Reactive Oxygen/Nitrogen Species (ROS/RNS)
•ROS/RNS are molecules are known to induce damage to important biomolecules:
•DNA
•Lipids
•Proteins
•Produced from both normal metabolic processes and from external sources.
Peroxynitrite
Superoxide
Hydrogen Peroxide
The Free Radical Theory of Aging
•With age, ROS/RNS increase
•Greater appearance
•Loss of antioxidant defenses
•Implicated in age-related diseases
•Atherosclerosis
•Cancer
•Macular Degeneration
•Neurodegenerative Diseases
Antioxidant Defenses Decline with Age
*Glutathione Disulfide (GSSG)
Oxidized by free radicals
Reduced by Glutathione Reductase
Example: Glutathione (GSH)
Glutathione (GSH) Most abundant low mol. wt. acqueous
soluble antioxidant Co-substrate for GSH S-transferases
and peroxidases Synthesized in all mammalian cells
Old
You
ng
0
20
40
60
Glu
tath
ion
e(n
mol/m
g p
rote
in)
• Found in Green Leafy Vegetables
• Chiral Molecule
• Natural form: R-Lipoic Acid
•Commercial Preparations are a 50:50% mixture of R- and S-Lipoic Acid
•Used as a Therapy for Hyperglycemia and Heavy Metal Poisoning
Asymmetric Carbon
Lipoic Acid : A Dietary Factor that Potentially Improves Antioxidant Defenses
Lipoic Acid Reverses the Age-Related Loss of GSH
Rats [young (3 mo) and old (24 mo)] were supplemented with diets containing 0.2% R-lipoic acid for two weeks prior to sacrifice and antioxidant analysis
Red
uce
d G
luta
thio
ne
(nm
ol/m
g p
rote
in) Young Old
Con
trol
+Li
poic
Aci
d
+Li
poic
Aci
d
Con
trol
0
20
40
60
80*P<0.03 vs. Young
*
Conclusions:• R-Lipoic Acid Improves GSH Levels in Aging rats after two weeks of supplementation
•Does Lipoic Acid improve Plasma GSH in elderly human subjects acutely and/or chronically?
•Is R-lipoic acid (the natural form) more effective than the racemic mixture in affecting Plasma GSH levels either acutely or chronically?
Key Questions:
19 Human Volunteers
Ten subjects (18-45 yrs old) Nine subjects (75+ yrs old)
• Volunteers fasted over night prior to taking 500 mg of R- or R,S-lipoic acid by mouth
• An indwelling catheter was placed in the cubita fossa and blood samples (3 ml) were taken over a 3 hour period
• Plasma glutathione (both reduced and oxidized) was measured by HPLC• In some samples, white blood cells were purified and glutathione levels
measured•Subjects acted as their own control
• Provided the other enantiomer of lipoic acid 1 week after the initial dose
Experimental Design
GSH/GSSG Quantification – High Performance Liquid Chromatography
•GSH and GSSG were derivatized with iodoacetic acid (IAA)•The IAA-GSH derivative was “tagged” with dansylchloride• Detected following HPLC separation by fluorescence monitoring
GSHInternal standard
GSSG
•Range: 0.4 to 3.2 µM GSH
•Young: 1.41µM ; Old: 2.31 µM
•No statistical differences between age groups
Baseline GSH Levels in Plasma of Young and Old Subjects
P=0.75P=0.9
Plasma GSH Levels Do Not Change Following an Acute Oral Dose of R- or R,S-Lipoic Acid
Conclusions
•Plasma Analysis of GSH from young and old subjects using HPLC with Fluorescence Detection gave values that agreed with previous studies
• A single R-LA dose caused a trend to increase plasma GSH values in youngSubjects (but not statistically significant)
• Plasma GSH levels from old subjects showed no changes over 3 hours
Future Plans• Increase the number of subjects analyzed for plasma GSH changes following an acute oral LA Dose
• Currently have ~60% of samples analyzed, run remaining samples to increase N-value
• Analyse GSH levels in White Blood Cells following administration of LA
• Determine whether Chronic LA supplementation increases plasma (and WBC) GSH levels in young and/or old subjects
Thank you to:
Dr. Tory Hagen
Judy Butler
Alan Taylor
The Hagen Lab
Dr. Kevin Ahern
Cripps Scholarship Fund
The HHMI program