Reactive Oxygen Species

Example: Asphyxiation, what will happen to blood pH

Decrease in blood pH because of low oxygen

Example: Asphyxiation, What happens to glycolysis

Glycolysis increases, anaerobic, end with lactic acidosis

Example: Asphyxiation, What happens to the TCA cycle

Inoperable, high NADH

Hypoxia

lack of oxygen

What does CO poisoning block

Blocks hemoglobin of the ETC and O2 usage, blocks oxidative phosphorylation

Hypoxia can be caused by what events

Acute anemia
Ischemia asphyxiation

What is glycolysis activated by in lactic acidosis

Low ATP

TCA cycle in lactic acidosis

Inoperable, high NADH

How is pyruvate diverted to lactate

Acetyl CoA accumulates; PDH shut down; pyruvate diverted to lactate

Where is lactate sent

To circulation

Question: What type of proteins are induced by hypoxia

Glycolytic

Example: Hypoxia during Ischemic cell injury, myocardial infarction

Blood clot in the coronary arteries
Stops blood supply to the heart
Often, part of the heart muscle dies
Depleted ATP, accumulated ADP and NADH
Blocks TCA cycle

ATP is critical for maintaining what

Ionic radiance of calcium, sodium, and potassium

What happens when you break down ionic radiance of Ca++, Na+, and K+

Bad osmolar things happening leading to cell-lysis, RBCs susceptible to this because of glycolysis dependency

Adaptation to Hypoxia: Induction of HIF

Regulated by levels of oxygen
Have oxygen sensors: prolyl hydroxylase (PH)
more O2 = more PH
Hydroxylate HIF-alpha
Eating up by Proteasome (destroyed)
(not destroyed) Goes to nucleus
Binds to HIF-Beta
Forms regulatory complex
Increase transcription of glycolytic enzymes

Where are prolyl hydroxylases important

HIF
Connective tissue
Collagen

How are mitochondrial myopathies present

Muscles myopathies (weakness)
Neurological sx due to impaired mitochondrial function

When do mitochondrial myopathies pop up

Spontaneously, since they have their own DNA

What does MELAS stand for

Mitochondrial myopathy, Encephalopathy, Lactic acidosis, and Stroke-like episodes

What does MERRF stand for

Myoclonic epilepsy and Ragged red fibers

MELAS and MERRF result from what

maternally inherited defects in genes encoding mitochondrial tRNAs

What are MELAS

Poorly performing mitochondria that don't produce enough ATP, affecting brain and muscle

Mutation rate of Mitochondrial DNA

Much faster than nuclear DNA, increasing chance of mitochondrial myopathies from mitochondrially encoded genes

Why is oxygen dangerous (ROS)

Forms free radicals that are highly reactive

superoxide

O2 -1

Hydroxyl radical

Worst molecule, Highly reactive, reacts with anything near by, short

3 enzymes critical for regulating ROS

Superoxide dismutase
Catalase
Glutathione Peroxidase

Superoxide damages

Damages biomolecules (lipids, proteins, DNA)
Forms other reactive species

What is one mechanism for the generation of superoxide

NADH Dehydrogenase, Complex 1

NADH Dehydrogenase, Complex 1

NADH. drops off its e- to FMNH2

Transfer e- to waste station, QH2 (1 at a time)

in between

FMNH radical

Free O2 will pick it up

Turn it into superoxide anion

What does free Iron catalyze

Hydroxyl Radical Formation: OH*

iron reduction by superoxide

Superoxide anion can reaction with ferric iron to form oxygen and ferrous iron

Fenton Reaction

Ferrous iron from superoxide reduction can do another reaction with H2O2

What does the fenton reaction produce

Hydroxyl radical: HO* and regenerates ferric iron

Haber-Weiss Rxn

Iron catalysis of hydroxyl radical formation from superoxide

Where is iron present in the body

Hemoglobin
ETC

Fe3+

Ferric form: most oxidized

Fe2+

Ferrous form: less oxidized

Example: Free radical oxidants (can form free radicals and other highly reactive products)

RO2* Peroxyl

RO- * Alkoxyl

H02* Hydroperoxyl

NO* Nitric oxide

Free radical damages: Proteins, where do they act

On free cystines, oxidation of sulfhydryl group on cys and Met side chains , adds a hydroxyl group, oxidizing it further

Free radical damages: Proteins, what do they disrupt

Iron sulfur centers in redox enzymes, breakage of peptide bonds

Free radical damages: DNA: Oxidation of bases

8-oxoguanine removed by base excision repair, double-strand breaks

Free radical damages: Lipids

Oxidation of polyunsaturated (not saturated or monounsaturated) fatty acids, causing rancidity

Free radical damages: Lipids Mechanism is what

Free-radical propagation

rancidity

Spoilage caused by breakdown of fats

Lipid Peroxidation: Malondialdehyde formation

Superoxide anion can come in
Polyunsaturated fat can hit C in between
Generates free radical
Cause bond breakage and oxidation
Generate Malondialdehyde

Malondialdehyde

Product of lipid oxidation that can crosslink proteins and DNA, highly reactive and destroy DNA function

Lipid Peroxidation: Free Radical Propagation Mechanism

Single free radical can cause multiple oxidations

Start with OH*

Generate radical: Lipid radical

Free O2 comes in

Modifies and forms peroxide: Lipid peroxyl radical

Transfers free e- to another lipid

Generates Lipid peroxide

Regenerating Lipid radical: Propagation

What does Lipid peroxide end up doing

Smelling bad, tasing bad, causing further breakdown of fatty acid chains

superoxide dismutase

catalyzes conversion of superoxide radicals to molecular oxygen and hydrogen peroxide

Catalase

breaks down hydrogen peroxide to molecular oxygen water

How many H2O2 are needed to generate 1 oxygen and 2 waters

2

Glutathione peroxidase

Can break down H2O2, uses 2 small molecules glutathione in a reduced form (2 G-SH) oxidizes to G-S-S-G and turns H2O2 into water

Glutathione is synthesized where

In all cells

Glutathione reduced is what

G-SH

Glutathione oxidized form is what

GS-SG

GS-SG disulfide bonds between cysteines

Chemically the same as disulfides in proteins

Glutathione serves to do what

Maintain cellular redox state

Where is the reducing environment: [GSH] > [GSSG]

Cytoplasm

GSH is regenerated by what

GSSG using NADPH reducing equivalents

Glutathione structure

Made of 3 amino acids
Not a peptide
Has SH from cysteine

Cytoplasmic NADPH/NADP+

Some irreversible reactions in the metabolic pathways reduce NADP+ to NADPH

How do cells maintain a high NADPH/NADP+ ratio

1. Glucose-phosphate-dehydrogenase (HMP OR PPP)
2. Malic enzyme (Malate to pyruvate)

Mitochondrial antioxidant defenses

transhydrogenase that is fueled by the proton gradient uses NADH to maintain a high NADPH/NADP+ ratio for the glutathione reductase reaction

G6PDH deficiency,

impairs the ability of an erythrocyte form NADPH, resulting in hemolysis

Clinical Correlation: Favism, is what

deficiency in the first enzyme in the hexose monophosphate shunt G6PDH

hemolysis

the rupture or destruction of red blood cells.

Favism is what type of defect

Partial, reduced enzymatic energy

Favism is RBCs can be from what

Oxidant stress
Fava beans
Certain drugs

What happens with oxidant stress in RBCs

Produce more H2O2

Glutathione peroxidase goes into action

Generate GS-SG

Need to regenerates, but not enough NADPH End up in hemolytic crisis in RBCs

400 million have this mutation

Clinical Correlation: ALS Amyotrophic Lateral Sclerosis

Neurodegenerative disorder, late onset with neural degeneration, loss of motor function and associated central neurons

Mutations in what is responsible for ALS

Mutations in superoxide dismutase

ALS results in what

Paralysis, fatal in 2-5 years

20% of familial ALS cases are associated with what

SOD 1 mutations (1-2% of all ALS cases are)

ALS is also known as what

Lou Gherig's disease

Main dietary antioxidants humans use

Ascorbic acid - Vitamin C

What is Vitamin C

natural antioxidant, high requirement of it, co factor is some enzymes (proyl hydroxylase)

Dietary antioxidants: Water-soluble

Ascorbic Acid - Vitamin C
Uric acid
Sulfhydryl compounds
Phytochemicals

Dietary antioxidants: Fat-soluble

Alpha-tocopherol (Vitamin E)
Retinoids (Vitamin A)
Carotenoids (Vitamin A)
Ubiquinone (CoQ, reduced)

Epidemiology

Diets high in antioxidants are associated with health benefits

intervention studies

No consistent benefits when a single antioxidant is given in medium to high doses

Antioxidants

Organic molecules that help protect the body from harmful chemicals called free radicals

Example: Antioxidants - Vitamin C

Vitamin C can take up e-

Form intermediate free radicals (stable)

Can take up several electrons

End as a stable product that can be excreted in the form of dehydroascorbic-acid

Example Role of NOX2: NADPH oxidase Phagocytosis

Immune system reacted to bacterial invasion

Binds to bacterium

Forms a phagosome, taking in bacterium

NADPH oxidase combines with oxygen, uses NADPH to form superoxide anions

Directly attack bacterium, destroy function of proteins, DNA, and lipids

OR

Can become H2O2 by superoxide dismutase

Myeloperoxidase

Generates hydrochloric acid

Generates hydroxyl radicals

Destroying bacteria

NADPH oxidase Phagocytosis does what

Generate free radicals on purpose during Phagocytosis in order to destroy ingested microorganisms that we want to get rid of

Response system to oxidative stress

Goes through a transcriptional regulatory mechanism

Antioxidant Defense System steps

Have a lot of SH groups

oxidants

Forms disulfides and stabilizes complex

KEAP1 and NRF2 separate

NRF2 goes into the nucleus

OR

No oxidation

SH groups

Ubiquitination

Takes it to the proteasome for degradation

TF NRF2 stimulates what

Transcription of antioxidant genes by binding to the antioxidant response element (ARE) in promoters and enhancers

What happens in the absence of oxidant stres

Kept in the cytoplasm and sent to the proteasome by KEAP1
Oxidants release KEAP1 from NRF2, allowing NRF2 to go to the nucleus and stimulate transcription

Regulated genes

Genes for enzymes of glutathione metabolism and NADPH production, genes for proteostasis proteins (proteasome, autophagy), genes for enzymes of phase 2 drug metabolism

What are 3 words for oxygen deprivation with serious consequences

Hypoxia
Anoxia
Ischemia

What regulates the levels of ROS

Antioxidants and the cellular ROS enzymes
SOD, Catalase, Glutathione peroxidase

Example: Non-radical oxidants

O3 Ozone

1O2 Singlet Oxygen

HOCI- Hypochlorous acid

ONOO- Peroxynitrite