See also Antioxidants  See also Oxidized and reduced variants in Antibody purificaiton. 

Oxidation and reduction are part of something in chemistry known as redox reactions which are chemical reactions in which atoms have their oxidation number changed. Redox reactions can be simple as with reduction of carbon by hydrogen to yield methane or they can involve very complex electron transfers as with oxidation of sugar in the human body.

Thus redox reactions involve both oxidation and reduction because one can not occur without the other. Redox reactions are essential in biology. For example, photosynthesis involves the reduction of CO2 into sugars and the oxidation of water into molecular oxygen. The reverse reaction, respiration oxidizes sugars to produce CO2 and water. As intermediate steps, the reduced carbon compounds are used to reduce NAD+ which then contributes to a proton gradient, which drives the synthesis of ATP and is maintained by the reduction of oxygen.

The term redox state is often used to characterize the balance of NAD+/NADH and NADP+/NADPH in a cell. An abnormal redox state can occur in a variety of situations such as sepsis and shock.

Oxidation: 

Oxidation involves the loss of an electron by a molecule, atom or ion. (ie., loss of H or gain or O). Oxidation is sometimes referred to as gaining a positive charge.  Oxidation is used in industry with the production of cleaning products.

Oxidizing Agents:

Substances which have the ability to oxidize other substances are known as oxidative or referred to as oxidizing agents, oxidizers and oxidants. Oxidants remove electrons from other substances. Thus oxidants are themeselves reduced. Examples of oxidants include H2O2, CrO3 and have high oxidation numbers. They are highly electronegative that can gain one or two extra electrons by oxidizing a substance.

Oxidizing agents, preferably oxidizing metal ions such as Cu2+, Fe2+, Fe3+ and Zn2+Schotte (US13/266503 and US2012/0157664).

Pathways Leading to reactive oxygen species (ROS):

Pathways leading to the generation of ROS/RNS from H2O2 include the following:

1st superoxide anion (O2-) is metabolized via the dismutation reaction to give hydrogen peroxide. This reaction is catalyzed by superoxide oxidoreductase dismutase (SOD), a cytoplasmic enzyme that is constitutively expressed, and by a mitochondrial enzyme that is induce in response to oxidant stress.

(1) H2O2 ——-nucleophilic reaction: H2O2 can be converted by one pathway involving iron into the hydroxyl radical (OH), an injurious ROS causing cellular damage. The nucleophilic reaction with H2O2 can be the oxidation reaction observed when protein product is exposed to vapor H2O2 used as aseptic agent or even from the degradation of commonly used excipients such as polysorbates (e.g., Tween) or polyethylene glycols. 

(2) H2O2 + Fe+2 (or Fe+3) —-OHo——-ROS: When trace metal (e.g., iron, copper, or chromium) is brought to the formulation solution, for example from contact with stainless steel, a Fentron reaction, H2O2 with Fe(II), becomes operative. This iron-catalyzed reaction is impeded by the iron chelator desferrioxamine, which is also capable of nuetralizing the toxicity of OH.

3) H2O2 — HOCL: In another reaction, H2O2 can be convereted to hypochlorous acid (HOCL) releasing singlet oxygen (O2) and chloride ions in the process.

4) another pathway diverges from O2- to generate nitric oxide (NO), peroxynitrite anion (OONO-), and peroxynitrous acid anion (HOONO). HOONO can be converted to OH thereby releasing nitrogen dioxide radical (NO2-)

The primary mechanism to form ROS is often referred to as a Fenton-type reaction, which is the catalytic reaction between hydrogen proxide and redox active metals (such as ferrous cations) to produce pwoerful oxidants that can oxidise a wide number of important biomolecules: (Tomas McKenna, “Oxidative stress on mammalian cell cultures during recombinant protein expression” Department of Physics, Chemistry and Biology, 2009).

Reduction: 

Reduction involves the uptake of an electron by a molecule, atom or ion (i.e., gain of H or loss of O). Reduction is also sometimes referred to as reducing an atom’s positive charge.

Reducing Agents:

A “reducing agent” refers to a compound which reduces molecuels in its environment, i.e., which changes molecles to become more reduced. A reducing agent acts by donating electrons, thereby becoming itself oxidized after having reduced a substrate. Examples of reducing agents include dithiothreitol (DTT), mercaptoethanol, cystein, thioglycolate, cysteamine, gltathione, and sodium borohydride. (Shuurman, WO 2008/119353). 

Substances which have the ability to reduce other substances are said to be reductive and are known as reductive agents or reducers. A reductant transfers electrons to another substances. Typical reductants are reduction electropositive elemental metal such as Li, Na, Mg, Fe, An, Al. These metals donate electrons readily.

Definitions

Free radical: is any chemical species capable of independent existence possessing one or more unpaired electrons. Free radicals are thus very unstable. Environmental stimuli such as ionizing radiation (from industry, sun exposure, cosmic rays and medical X-rays), environmental toxins can increase the elvels of free radicals. Lifestyle stressors such as cigarette smokine and excessive alcohol consumption can also do this.

Radical species may combine to form other more damaging or toxic species such as peroxynitrite (O=NOO-).

Free radicals react with key organic substrates such as lipids, proteins, and DNA. Oxidation of these biomolcules can damagage them and disturbe normal cell functioning.

Compounds which Play Role in Oxidation/Reduction

Reactive Oxygen Species (ROS): is a term collectively describing radicals and other non-radical reactive oxygen derivatives. These intermediates may participate in reactions giving rise to free radicals or that are damaging to organic substrates. ROS in living organisms include the following radicals: Hydroxyl (OH), superoxide (O2-), nitric oxide (NO), Thyl (RS), peroxyl RO2, lipid peroxyl LOO. Non-radicals include hydrogen peroxice H2O2, ozone (O3), hpochloric acid (HOCL)

Oxidative stress: occurs when the generation of ROS in a system exceeds the system’s ability to neutralize and eliminate them. If not regulated, the excess ROS can damage a cell’s lipids, protein or DNA, inhibiting normal function.

Aerobic organisms use oxygen O2 as a terminal electron acceptor in the mitochondrial resperiation pathway (oxidative phosphorylation), which is the cells greatest source of oxygen free radicals under normal conditions. O2 -(electron transfer)-O2–(electron transfer)—H2O2–(electron transfer) -OH—(electron transfer)–H2O. One to two percent of the oxygen consumed by a cell may be transformed into oxygen radicals, which then lead to the production of reactive oxygen species (ROS). ROS appear to be the most common cause of most degeenrative eukaryotic cell disorders. Free radicals can cause single and double strand breakage in DNA, inappropriate cross-oinking in proteins and even oxidation of lipids. As specific growth rate and resperiation levels rise, so too may the leves of oxygen radicals rise, causing damage to the cell. The primary mechanism to form these reactive intermediates is often referred to as a Fenton-type reaction (see above), which is the catalytic reaction between hydrogen peroxide and redox active metals (such as ferrous cations) to produce pwoerful oxidants that can oxidise a wide number of important biomeolcules. (Tomas McKenna, “Oxidative stress on mammalian cell cultures during recombinant protein expression” Department of Physics, Chemistry and Biology, 2009). 

What Effects Intracellular Redox Balance in Cells:

Antioxidants: Antioxidants have been shown to downregulate cytokine transcription and biosynthesis. A collection of antioxidants for all injectable products was compiled by Neema (J Pharm Sci Tech 51: 166-71 (1997).

Free methionine has been routinely used as an antioxidant. However, this amino acid alone does not protect against all mechanisms of oxidation and is most effective in inhibiting nucleophilic oxidation of methionine or cysteine residues.

Role of GSH: In physiological conditions, the intracellualre redox status of thiols is highly reductive. The tripeptide of L-y-glutamyl-L-cysteinyl-glycine, or glutatione (GSH) a buiquitous thiol, plays a major role in maintaining intracellular redox balance and regulating pathways augmented by oxidative stress. GSH, for example, is present in high concentrations in lung epithelial lining fluid and has been reported to maintain the integrity of the airspace epithelium in vitro and in vivo.

Gluthatione depletion is associated with the augmentation of a pro-inflammatory signal by up-regualting ROS.

Cytokines: are mediators of oxidative stress and have the potential to alter redox equilibrium. IL-1 induced responses in mesangial cells, for instance, occurs through modulating redox equilibrium.

Studies have shown that various antioxidant enzyme genes including manganese-dependent SOD, metallothionein, glutatione S-Transferase, and ferritin heavy chain (fhc) are induced by TNFalpha in an NF-kB dependent fashion.

Effects of Redox Balance on Cytokine Production:

ROS: ROS signaling regulating the transcription of IL-4, IL-6, IL-8 and TNFalpha ini several cells models occurred through a thiol-dependent mechanisms.

H2O2 generates the OH radical, induces in a dose dependent manner the release of IL-1beta, but to a lesser extent IL-6 and TNF-alpha.