Introduction to ROS and ROI
Aaron Bissell
What are ROS and ROI?
Reactive oxygen intermediates or ROI are successive 1 e- reduction products of diatomic oxygen en route to the production of water (Figure 2.). As you can see in the Figure 1 below, there are many different ROI known at the moment. In figure 2, Eq.1 is catalyzed by Superoxide dismutase (SOD). This reaction converts two extremely reactive superoxide anion to a more manageable hydrogen peroxide. Eq.2 is catalyzed by catalase and converts two hydrogen peroxides produced in reaction 1 and produces two moles of water and a diatomic oxygen. This allows the cell to reduce the number of ROI in the cell.
Figure 1 Reactive Oxygen Intermediates examples.
Figure 2. ROI reaction pathway from superoxide anion to water
A common misconception is that ROI and ROS (reactive oxygen species) can be used interchangeably. While all ROI fit within the category of an ROS, ROS are more broad and consist of external molecules such as ozone and singlet oxygen.
Sources
Internal sources
When talking about ROS and ROI, it is typically assumed that we are discussing within an organism, more specifically a human. In a human, ROI are produced in several places throughout the cell. As However the greatest production of this lies within the mitochondria and is a byproduct of the ETC.
Figure 3. Sources, breakdown, and interactions of ROS/ROI in the cell
External sources
While ROI/ROS are produced in the cell itself, they can also be produced by external sources. Three common external ROS sources are uv radiation, smoke inhalation, and pollutants. All three of these damage the cell, and speed up aging in the body.
ETC and ROI production
Within the cell the ETC is the greatest contributor to cellular ROI production. The ETC's job is to create ATP, while passing electrons through its complex. When it is normally working, as shown by figure 4, the ETC will use the electrons passed down the chain to produce water from diatomic oxygen and hydrogen ions. However roughly 1 to 2% of the electrons that are passed in this reaction will prematurely react with the diatomic oxygen to produce the highly reactive superoxide anion. This production of ROI is produce by two main factors both shown in figure 5. The first is due to an imbalance in the NADH/NAD+ equilibrium and the second is due to high concentration of the reduced form of CoQ and a drop in pressure.
Figure 4. Normal functioning ETC
Figure 5. Production of ROI in the ETC
Antioxidants
Within our cells we have ways to combat ROI other than SOD and Catalase using chemicals called antioxidants. These chemicals prevent or slow cell damage, which is extremely important for cell longevity. However antioxidants aren't actually a specific substance, it is the description of the ability for a molecule to be able to reduce ROI in the body. These are found in our day to day diet, along with over the counter supplements.
Figure 6. Food sources of different antioxidants
Oxidative stress balance
As with anything in our cells, equilibrium is important to maintain. ROS/antioxidant equilibrium is no different, and it important to maintain this to minimize cell damage due to oxidative stress. As you can see below in the scales on the right ROS concentration outweighs the concentration of the antioxidants, while in the scales to the left ROS concentration is equal to antioxidant concentration. It is important to note that it is not possible for the scales to shift to the antioxidant side. This is due to the fact that when antioxidants reduce ROS's they in turn become reactive radicals themselves, thus the need of antioxidants is always there.
Figure 7. Oxidative stress equilibrium illustration
Methods and techniques
While many different probes are used to assess ROS in the cell, the general method is the same. Due to the similarity in chemical reactions ROS molecules interact in, detecting specific ROS in the cell are difficult to run. The most common techniques used are fluorescent probes and chemiluminescent probes to tag the molecule and assess for its presence. One of the most specific tests using this methodology is the detection of extracellular hydrogen peroxide and uses N-acetyl-3,7-dihydroxyphenoaxazine or amplex red. This can be seen by figure 8 below.
Figure 8. Amplex red assay detection of extracellular hydrogen peroxide
