Antioxidants & Free radicals

What are Reactive Oxygen Species?  ROS also known as Free oxygen radicals  Any molecule with an unpaired electron  Extremely chemically reactive  Damage cell membranes  Responsible for more than 100 human diseases  Aging, cancer, heart attacks, stroke and arthritis  Some beneficial effects

How are ROS Formed?  Primary source is our body during energy production  Environmental contaminants  Ionizing and ultraviolet radiation  Prolonged low blood flow states (atherosclerosis, heart attacks and stroke)  Diet (fatty and processed foods)  Low levels of antioxidants

Protection from ROS Damage  Superoxide dismutase  Catalase  Glutathion  Antioxidants in diet  Supplementation

Aging and Effect on Antioxidant Enzymes  Significant decline in SOD  Significant decline in catalase and glutathione  Significant decline in energy production  Cellular, tissue and system aging and failure

Scientific Support for Antioxidants  Animals with longer life spans have higher antioxidant levels  Dietary increase in antioxidants increase life span  Caloric restriction (reduces ROS formation) leads to significantly increased life span

Questions asked  What are free radicals?  Types of free radicals  Sources of free radicals  Oxygen metabolism

 Discussion on oxidative damage and oxidative stress  What is antioxidant?  Natural free radical defense systems  What are Phytochemicals?

Nutrients Non-nutrients Energy, building materials Factors regulating metabolism metabolism LipidProteinCarbo-hydrateVitaminsMinerals EFA & non-EFA Glucose Water Phyto- chemicals chemicals pigmentspigments AntioxidantsAntioxidantsFibers Other food components Food GSHCys Functional food or Neutraceuticals C, E,  -Car Zn, Se EAA & non-EAA

Free radical-Mediated Diseases  Diseases of the old - Chronic and degenerative diseases (diabetes, cataracts, Alzheimer ’ s disease, cancers, cardiovascular disease, and aging)  Diseases of the young and innocent - Acute and immature diseases ( Eyes: retinopathy of prematurity, Lung: bronchopulmonary displasia, Brain: cerebral pulsy, Pancreas: Type 1 diabetes)

What are free radicals?  Any molecule containing one or more unpaired electrons

Types of Free Radicals  Reactive Oxygen Species (ROS) -  Reactive Nitrogen Species (RNS) - NO.  Reactive Metabolites or Intermediates - metabolic activation of drugs, toxins, pollutants, cigarette smokes, etc.

Reactive Oxygen Species (ROS)  Superoxide (O 2. -)  Hydrogen Peroxide (H 2 O 2 )  Hydroxyl Radical (OH. ) - product of Fenton reaction catalyzed by free Fe and Cu  Singlet Oxygen ( 1  g O 2 ) - oxygen at an excited state, requiring photosensitizers and photons

Sources of oxygen free radicals  In mitochondria: - generation of energy - ATP - glucose, fatty acids, amino acids - O 2 2H 2 O 4e - - leakage of O 2 -. (superoxide) H 2 O 2 (hydrogen peroxide)

 In Smooth Endoplasmic Reticulum (microsome) - detoxification (cytochrome P-450s) - toxins, drugs and xenobiotics - O 2 + RH R-OH and H 2 O - leakage of O metabolic activation - X.

 In Peroxisomes - containing oxidases for degradation of various substrates - glucose, amino acids, xanthine, etc. - requires O 2 - byproduct is H 2 O 2

 In Cytoplasm - nitric oxide (NO. ) production from Arginine - functions as a biological messenger - in brain, vascular endothelial cells, and macrophages - NO. + O 2 -. ONOO. (peroxynitrite)

NO: a Biological Messenger  NO is a neurotransmitter (brain- bNOS)  NO regulates blood pressure (vascular endothelial cells- eNOS)  NO is a cytotoxic agent (macrophages- iNOS)

 Production of Singlet Oxygen - photosensitizers in the biological system (bilirubin, riboflavin, retinal, porphyrin) - requires light, O 2 and photosensitizers - chlorophyll in photosynthesis - photodynamic therapy

Antioxidants  Prevents the transfer of electron from O 2 to organic molecules  Stabilizes free radicals  Terminates free radical reactions

Free Radical Defense System  Antioxidant Enzymes  Antioxidant Quenchers  Antioxidant from Foods – nutrients/non- nutrients

Antioxidant Enzymes  Superoxide Dismutase (SOD) – to get rid of superoxide produced from electron transport chain, the product is hydrogen peroxide.  MnSOD (mitochondria).  CuZn SOD (cytosol).

Oxygen Radical Defense Enzymes O2¯O2¯H2O2H2O2 H 2 O + O 2 Mn SOD Catalase GSH Peroxidase CuZnSOD OH Fe 2+

Antioxidant Enzymes - 2  Glutathione Peroxidase (GSH PX) – to get rid of hydrogen peroxide (H 2 O 2 ) and some lipid peroxide. It requires reduced glutathione (GSH) as substrate and produces oxidized glutathione (GSSG) as product. A cytosolic enzyme.

Functions of GSH-dependent Enzymes L-OH H 2 O GSSG NADPH L-OOH H 2 O 2 GSH NADP+ X.X.X.X. GSX X -Mercapturic Acid GSH Px GSH-TR GSH--Rx

Glutathione  GSH is a tripeptide,  -glutamyl- cysteinyl-glycine  The sulfur atom of the cysteine moiety is the reactive site which provides electrons  GSH is stable because the  bond in glutamyl-cysteine (not the  peptide bond) is resistant to cellular peptidases

Glutathione  GSH is the most abundant non-protein thiol in mammalian cells  GSH is a substrate for two enzymes that are responsible for detoxification and antioxidation.  Other physiological roles including cysteine storage and transport, prostaglandin metabolism, immune function, cell proliferation and redox balance

Glutathione Synthesis Protein Protein Methionine Cysteine Glutamate  -Glutamylcysteine Glycine

Antioxidant Enzymes - 3  Catalase – to get rid of hydrogen peroxide produced in peroxisome.

Antioxidant Quenchers  Cellular proteins which chelate pro-oxidant minerals (iron and copper or others)  Transferrin – iron transport protein  Ferritin – iron storage protein  Metallothionein – minerals and heavy metals (Zn/Cu/Cd/Hg)  Ceruloplasmin – copper transport and storage

Antioxidants From Food  Antioxidant nutrients – vitamin E, vitamin C, (vitamin A?), beta-carotene  Phytochemicals – antioxidants from plants

Discovery of Other Functions of Phytochemicals  Anti-oxidant  Anti- inflammatory  Anti-estrogenic  Anti-allergic  Anti- cholesterolemic  Anti-hemorrhagic  Anti-mutagenic  Anti-neoplastic