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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
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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. Superoxide Peroxynitrite 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
60 40 20 0 Antioxidant Defenses Decline with Age Example: Glutathione (GSH) Oxidized by free radicals Reduced by Glutathione Reductase *Glutathione Disulfide (GSSG) • Glutathione (GSH) • Most abundant low mol. wt. acqueous soluble antioxidant • Co-substrate for GSH S-transferases and peroxidases • Synthesized in all mammalian cells (nmol/mg protein) Glutathione Young Old
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
80 60 40 20 0 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 *P<0.03 vs. Young Young Old Reduced Glutathione (nmol/mg protein) * +Lipoic Acid +Lipoic Acid Control Control • 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=0.75 P=0.9
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