Antioxidants and Aging: The Role of Glutathione

Presented by: Joanna Helm Masters of Clinical Nutrition Candidate Graduate Programs in Human Nutrition

 74 years young, gonna keep on having fun

Buddy Guy

Learning Objectives

1. Describe the role glutathione plays as an antioxidant in the body

2. Identify the diseases of aging associated with glutathione deficiency 3. Explain the risks and benefits of glutathione supplementation in the elderly population

Balance

Free Radicals
Unpaired electron

Electron donation

Nucleus

Nucleus

Antioxidant

Free Radical

Free Radicals

Antioxidant System
Catalase Peroxidase

OH
Hydroxyl radical
OH-

Superoxide Dismutase

O O
Peroxide
-O2-2

VS

Glutatione Peroxidase (Gpx)

Glutathione Reductase
(Grx) Vitamin C Vitamin E

O O
Superoxide anion
.O
2 Sies 1997

LEO says GER

Lose Electron Oxidation Gain Electron Reduction

What are environmental sources of free radicals?

http://www.medicinaosteopatica.it/public/44_2.jpg

Free Radical Theory of Aging

Introduced in 1956 by Denham Harman, MD, PhD Proposed the concept of free radicals role in the aging process
Damage Accumulation Theories free radical theory glycation theory error catastrophe theory membrane theory entrophy theory Genetic Theories Programmed to a set life span
Harman, 1956

Damage begins with our first breath

Cadenas & Davies 2000, Valko 2007

Glutathione (GSH)

Are these essential or non essential amino acids?

Braganza, 2010

Absorption
Lumen or Blood
Glutathione
Glutamate
Amino acid transporter

Cytoplasm

Blood

Glutamate

Cysteine
Amino acid transporter

Cysteine

Glycine
Glycine
Amino acid transporter

Glutathione

Biochemistry

Reduced Glutathione (2 GSH)

NADP+

Hydrogen Peroxide H2O2

Riboflavin

Selenium GSH peroxidase

GSH reductase

Water
NADPH + H+

Oxidized Glutathione (GSSH)

Rebrin, 2008

What happens if your body does not make glutathione?

1. Liver would fail with no way to excrete pollutants, drugs, or toxic metals Cells die from due to the impact from free radical damage Immune system would be vulnerable to bacteria, viruses and disease

2.

3.

Board 2013, Valko 2007

Aging, Illness and Glutathione Depletion

LHP Lipid hydroperoxide GSH - Glutathione

8.51

8.84

Severity of illness
2.14

3.14

Oxidative markers (LHP)

0.54

Decrease in plasma glutathione Result = reduced ability to manage oxidative stress

p=.0001

0.29 0.24 0.17

Young, healthy
( 24.5 +/- 4.7 yrs)

Elderly, healthy
( 70.7 +/- 4.8 yrs)

Elderly, chronic
(75.7 +/- 8.3 yrs)

Elderly, inpatient
(77.2 +/- 8.6 yrs)

n=66

n=58

n=49

n=47

Nuttal, Lancet, 1998

Young vs Old Glutathione Concentrations

Mean (+/-SEM) glutathione concentrations in healthy young (n=8) and healthy elderly (n=8) subjects before and after supplementation with cysteine & glycine GSH deficiency in the elderly occurs due to diminished supply of precursors, not due to a diminished capacity to synthesize GSH. a
2.08
+/- 0.12

c b
1.12
+/- 0.18

94.6%

2.18
+/- 0.35

Young

Elderly presupplement

Elderly postsupplement

p<0.05 on the basis of 1 factor ANOVA and Bonferroni-corrected t-tests

a b C

Significantly different from elderly-pre supplementation (b) Significantly different from elderly-post supplementation (c) Not significantly different from young (a)

Sekhar 2011

Cataracts
Development of Cataracts
Presence of Free Radicals in lens

Wild type mice with Glutathione Peroxidase activity

Crystalline fibers in the lens cannot repair damaged proteins Accumulation of proteins at the center of the lens

Knock-out mice NO Glutathione Peroxidase activity

6 months = ~25-30 human years

Protein build up allows more UV light to enter the eye

Exacerbates the cycle of oxidative damage

Fan 2012

Cataracts
Impact of Glutathione Peroxidase deficiency on cataract formation in the mouse lens
GPX Knockout mice

50% had cataracts by 4 months of age

20% developed into nuclear cataracts by 9 months Every mouse had over 60% lens opacification where wild type mice had less than 20%
LEGSKO mice were genetically altered to not produce Glutathione Peroxidase. Slit-lamp images were taken periodically and lens opacity/cataract of any size were recorded for 9 months in homozygous LEGSKO mice vs. age matched wild type mice (n = 30 mice per group).

Fan 2012

Cancer therapies
1) Pro-drug is inactive when administered. 2) Taken into the cell and binds to glutathione (purple molecule). 3) Activation of the cytotoxic unit (orange), signals the oxidative stress pathway and apoptosis of the cancer cell. 4) Synergistic function with cancer cisplatins due to prodrug (blue) blocking the ability of the cisplatin to bind. (Cisplatin binding to glutathione creating drug resistance)

Telynta & Telcinta Currently in Phase II and Phase III clinical trials
Ortega 2011

N-acetyl cysteine (NAC)

Cysteine precursor Well absorbed in intestine No effect if GSH is within normal range Recommend 600 mg / day as supplement DO NOT supplement with nitroglycerin Easily purchased online, GNC, Vitamin Shoppe
Cost range: $14-$30/bottle, $0.02-$0.50/capsule
http://reference.medscape.com/drug/n-acetylcysteine-mucomyst-acetylcysteine-343425#90

Immune Deficiencies
N-acetylcysteine supplementation in postmenopausal women

* * *

* * * * *

*
*

n=18

n=18

n=18

n=18

n=18

n=18

Each column represents the mean +/- SE of duplicate assays *p< 0.01 Arranz, 2008

Cognition and Neurodegenerative Diseases

Excitatory amino acid transporter (EAAT) dysfunction

Cysteine uptake

Glutathione synthesis

Oxidative Stress Aging

Neurodegenerative Disease
Aoyama 2012; Currais, 2013; Pocernich 2012; Emir 2011; Huang 2010; Fu 2006

Young and at risk

Apo E 3/3 = lowest risk; n=21 Apo E 3/4 = intermediate risk; n=17 Apo E 4/4 = highest risk n=16
n= 25 age matched controls n= 58 with Alzheimers (AD)
*p<0.05

GCLC glutamylcysteine ligase GCLM glutamylcysteine ligase modifier GPx glutathione peroxidase Badia, 2013

*p<0.05 compared to Apo E 3/3

GSH synthesis

Values are means +/- SD

Dietary Friends of Glutathione

150 g of Glutathione from food Vitamin C Recommended supplement is 500 mg/day Methionine
Recommended supplement is 1600 mg/day

Selenium Copper B vitamins Zinc, Iron, Magnesium

Luschak 2012

DO NOT supplement L - CYSTEINE

Unsafe for routine oral administration Readily oxidizes to potentially toxic degradation products including hydroxyl radical Excitotoxin to the brain- similar to glutamate and aspartate

Toxic to the retina

Drug Interactions

Acetominophen depletes GSH

May cause liver failure in alcoholics

Ethacrynic acid diuretics, depletes GSH

Exubera- inhaled insulin action is
increased with NAC

Optimal Ratio of GSH:GSSG

Intracellular concentrations of GSH ranges from 0.1 15 /M Equilibrium between GSH and GSSG calculated using Nernst equation Highly controversial topic in Glutathione research
Compartment Cytosol Mitochondrial matrix Estimated pH 7.0 7.4 7.0 7.0 7.4 GSH:GSSG 3250:1 10-14:1 250:1 3:1 or 5:1 8:1

Mitochondrial inner membrane space

Endoplasmic Reticulum Plasma

Deponte, 2013

What labs can be used to measure oxidative stress?

Direct measure in red blood cells (RBCs)
GSH:GSSH ratio through enzyme assays and Nernst equation Fluorescent heme degradation products

Inflammatory markers IL-1, IL-6, TNF-alpha Lipids F2 Isoprostanes Protein carbonyls

Assays used in research to measure GSH

Gas chromatography-mass spectometry (GCMS) High pressure liquid chromatography (HPLC) ion exchange or fluorescence Trolox Equivalent Antioxidant Capacity Colorimetry Folin-Ciocalteu Reagent (FRC)

Clinical Pearls
Do they have a chronic condition? If so, how long have they been living with it? How balanced is their diet? What kind of medications are they taking? Are they over the age of 50? What other stressors do they have in their life? (Recent divorce, lost job, move, financial struggles, new baby, etc)

Thank you

Joanna Helm
helmj@ohsu.edu This presentation is available at www.harvestyourfood.com