1. Changes in Acute Glutathione Levels in Human Plasma Following Lipoic Acid Supplementation Shawn Johnson Dr. Tory Hagen

2. 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

3. 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

4. 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 Young 0 20 40 60 Glutathione (nmol/mg protein)

5. 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

6. 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 Reduced Glutathione (nmol/mg protein) Young Old Control +Lipoic Acid Control 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

7. 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:

8. 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

9. 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

10. 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

11. P=0.75 P=0.9 Plasma GSH Levels Do Not Change Following an Acute Oral Dose of R- or R,S-Lipoic Acid

12. 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

13. 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

14. Thank you to: Dr. Tory Hagen Judy Butler Alan Taylor The Hagen Lab Dr. Kevin Ahern Cripps Scholarship Fund The HHMI program