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
Superoxide
•Lipids
•Proteins
Peroxynitrite
•Produced from both normal metabolic
processes and from external sources.
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
Example: Glutathione (GSH)
Oxidized by free radicals
Reduced by Glutathione
Reductase
*Glutathione Disulfide
(GSSG)
40
 Glutathione (GSH)

0

Old
20
Young
(nmol/mg protein)
Glutathione
60

Most abundant low mol. wt. acqueous
soluble antioxidant
Co-substrate for GSH S-transferases
and peroxidases
Synthesized in all mammalian cells
Lipoic Acid : A Dietary Factor that Potentially
Improves Antioxidant Defenses
Asymmetric
Carbon
• 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
Young
Old
Control
+Lipoic Acid
*P<0.03 vs. Young
Control
+Lipoic Acid
(nmol/mg protein)
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
Reduced Glutathione
Lipoic Acid Reverses the Age-Related Loss of
GSH
80
60
40
20
0
*
Conclusions:
• R-Lipoic Acid Improves GSH Levels in Aging rats after two weeks of
supplementation
Key Questions:
•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?
Experimental Design
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
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
GSH
Internal
standard
GSSG
Baseline GSH Levels in Plasma of Young
and Old Subjects
•Range: 0.4 to 3.2 µM GSH
•Young: 1.41µM ; Old: 2.31 µM
•No statistical differences between age
groups
Plasma GSH Levels Do Not Change Following
an Acute Oral Dose of R- or R,S-Lipoic Acid
P=0.9
P=0.75
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 young
Subjects (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