changes in acute glutathione levels in human plasma following lipoic acid supplementation shawn...

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