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