By Eben van Tonder, 2 August 2025

Introduction

Ageing is a decline in the body’s capacity to manage its internal redox environment. At its biochemical core lies a paradox: oxygen, the molecule that sustains life, is also the primary driver of molecular degradation. This redox imbalance, manifested as oxidative stress, disrupts mitochondrial efficiency, damages cellular structures, and accelerates biological entropy. Central to this process is the interplay between electron management, nitric oxide signalling, free radical formation, and the body’s antioxidant defences.

This article builds upon the foundational work “Nitric Oxide and the Physiology of Ageing” (EarthwormExpress, 2024). We explore free radical formation and mitigation from a systems biology standpoint, with particular attention to nitric oxide’s dual role as both regulator and radical, and examine how antioxidant micronutrients, especially from plant sources, participate in a co-evolved redox symbiosis with human metabolism.

ATP and Electron Flow: The Architecture of Energy Production

ATP (adenosine triphosphate) is the primary energy currency of the cell. It is a small molecule made of three main parts: adenine (a nitrogenous base), ribose (a five-carbon sugar), and three phosphate groups linked in a chain. The bonds between the phosphate groups, especially the last two, store potential energy. When a cell needs energy to perform work such as muscle contraction, nerve signalling, protein synthesis, or transporting molecules, it breaks the bond between the second and third phosphate groups in ATP. This releases energy and converts ATP into ADP (adenosine diphosphate) and an inorganic phosphate (Pi). Cells regenerate ATP continuously through processes like cellular respiration, where glucose and oxygen are used to recharge ADP back into ATP. In this way, ATP acts like a rechargeable battery that powers nearly every function in living organisms.

Reduced cofactors NADH and FADH₂, produced in the TCA (tricarboxylic acid) cycle, are molecules that carry high-energy electrons and deliver them to Complexes I and II of the mitochondrial electron transport chain. The term cofactor refers to a substance that helps an enzyme perform its function; in this case, NADH and FADH₂ are cofactors to enzymes involved in cellular respiration. A factor simply means something that contributes to a process, and a cofactor is one that works alongside an enzyme. These reduced cofactors act as electron donors, enabling the chain of reactions that produce ATP. Once the electrons are transferred from NADH and FADH₂, they move down an electrochemical gradient through Complexes III and IV. This flow of electrons is harnessed to pump protons (H⁺ ions) across the mitochondrial membrane, building a proton gradient. Finally, the electrons reduce molecular oxygen (O₂) to water. The proton gradient powers ATP synthase, which uses the flow of protons back into the mitochondrial matrix to convert ADP into ATP. This entire system links electron transfer, oxygen consumption, and ATP synthesis in one elegant biochemical cycle (Mitchell, 1961).

However, a small proportion (estimated 0.1–2%) of electrons escape from complexes I and III, prematurely reacting with oxygen to form superoxide (O₂•⁻), a primary reactive oxygen species (ROS) (Brand, 2010). This leakage is not accidental but it increases with mitochondrial membrane potential, cellular age, and oxidative damage, creating a feedback loop of inefficiency and radical accumulation.

O₂ doesn’t “want” the extra electron. It gets it by accident when the flow of electrons in the mitochondria becomes inefficient. During normal energy production, oxygen is supposed to receive four electrons and combine with protons to form water. But sometimes, especially when mitochondria are damaged, overworked, or under stress, electrons leak out early before they reach the end of the chain. When this happens, oxygen picks up just one electron instead of four. This incomplete reaction turns it into superoxide (O₂•⁻), a reactive free radical. This accidental process is the main way superoxide is formed in the body and marks the beginning of many oxidative stress cascades that can damage cells, especially as we age.

We have said that electron leakage is not accidental and that it is not only part of our normal metabolism and beneficial and yet, when describing how superoxides are formed, that O₂ doesn’t “want” the extra electron. How can both be true or is this a mistake? 

The answer is yes to both. What turns the formation of superoxide from normal and necessary to harmful is the degree to which it is produced. This relates directly to the question of why it forms whether as a regulated by-product of normal metabolism or as an unregulated consequence of electron leakage under stress or dysfunction.

Superoxides are formed naturally and continuously in the body. It is not an accidental anomaly, but rather an inevitable by product of how aerobic life produces energy. In the mitochondria, the process of oxidative phosphorylation uses the electron transport chain to transfer electrons from nutrients to oxygen, generating ATP. Ideally, oxygen receives four electrons and is fully reduced to water. However, because this system is not perfectly efficient, a small fraction of electrons, typically about 0.1 to 2 percent, leak prematurely from Complexes I and III and reduce oxygen partially, forming superoxide (O₂•⁻).

This low level production of superoxide is not inherently harmful. In fact, it plays several physiological roles, including redox signalling, regulation of gene expression, and triggering adaptive stress responses. At minute concentrations, superoxide is harmless and often beneficial, especially when it is promptly converted into less reactive molecules like hydrogen peroxide by superoxide dismutase (SOD), and then further detoxified by catalase or glutathione peroxidase.

The danger arises when superoxide production exceeds the capacity of these antioxidant systems. This typically happens under conditions of metabolic overload, inflammation, ageing, hypoxia, or mitochondrial dysfunction, all of which increase electron leakage. When this happens, superoxide accumulates and begins to interact with other molecules in uncontrolled ways, for example, reacting with nitric oxide to form peroxynitrite, or contributing to the production of hydroxyl radicals via hydrogen peroxide and Fenton chemistry.

So, to clarify:
Superoxide formation is natural because it is an unavoidable result of oxygen based energy production.
It is not harmful in low quantities and even has regulatory functions.
It becomes harmful when its formation becomes excessive or unregulated, overwhelming the body’s antioxidant defences and initiating oxidative stress cascades.
The term “accidental” is used not to imply that its existence is a mistake, but rather to describe the unintended amplification of its concentration and reactivity under certain conditions.

This reflects a core principle in biology: many of the body’s essential processes, such as oxygen metabolism, come with built in risks. Life manages these risks through evolutionarily refined defence systems, until those systems begin to falter, as they often do with age.

Nutrients are essential in this entire system because they act as the body’s primary source of electrons. That is, their value lies not only in supplying carbon, nitrogen, or calories, but in their ability to donate electrons through enzymatic pathways. Different nutrients have different redox potentials, meaning they differ in how readily they give up electrons. Some donate electrons to generate ATP, while others serve in antioxidant defence, donating electrons to neutralise radicals and regenerate protective molecules like glutathione or vitamin C. In this sense, nutrients are not just fuel in the traditional caloric sense, they are electron fuel, and the flow of electrons from nutrients to oxygen is the central energetic transaction of life.

Examples include glucose, fatty acids, and amino acids, which donate electrons via NADH and FADH₂ to the mitochondrial respiratory chain. Antioxidant nutrients such as vitamin C, vitamin E, selenium, and polyphenols also play this role in a protective context, donating electrons to neutralise free radicals and repair oxidative damage. The body’s entire energetic and defensive infrastructure depends on this finely tuned exchange of electrons, sourced from nutrients and distributed with precision, until imbalance occurs and damage begins to accumulate.

What Is a Radical? Molecular Instability and Electron Theft

A radical is any atom or molecule that contains at least one unpaired electron in its outer orbital. This unpaired electron gives the molecule a high degree of instability and reactivity. In order to stabilise itself, a radical will seek to acquire an electron from a nearby molecule. In doing so, it often damages the target molecule, creating a new radical in the process. This initiates a chain reaction that can propagate across membranes, DNA, and proteins. Radicals are therefore highly disruptive agents in the cell’s biochemical environment. Because they are generated as natural by-products of cellular metabolism, particularly during oxygen-based energy production, cells have evolved antioxidant systems to control their levels. When this balance is lost, radicals accumulate and trigger oxidative stress, a central factor in ageing and disease.

Superoxide: The First Spark in Oxidative Chain Reactions

Superoxide (O₂•⁻) is often the first radical formed in the body’s oxygen metabolism. It is generated when molecular oxygen gains one extra electron, typically a result of premature electron leakage from mitochondrial respiratory complexes, especially Complexes I and III. We have already discussed how this happens during mitochondrial respiration under conditions such as ageing, inflammation, or high metabolic activity. The electron transport chain is designed to deliver four electrons to oxygen to form water, but when only one is transferred, superoxide is formed. Because it contains one unpaired electron, superoxide is highly reactive and initiates oxidative chain reactions by transferring or donating that electron to other molecules.

Although superoxide itself is moderately reactive, its real danger lies in what it leads to. It can react with nitric oxide to form peroxynitrite, or it can be converted by the enzyme superoxide dismutase into hydrogen peroxide, which in turn can give rise to hydroxyl radicals through the Fenton reaction. These processes tend to affect tissues that use a lot of oxygen such as the heart, brain, liver, and skeletal muscle, and in cells with impaired antioxidant defences. It can oxidise membrane lipids, denature proteins, damage mitochondrial DNA, and accelerate the onset of cellular ageing, senescence, and programmed cell death.

Hydroxyl Radical: The Wildfire of Oxidative Stress

Hydroxyl radicals (•OH) are among the most destructive reactive species known in biological systems. Unlike superoxide or hydrogen peroxide, which can be detoxified or compartmentalised, the hydroxyl radical reacts almost instantly with whatever molecule is closest, lipids, proteins, carbohydrates, or DNA. It has no enzymatic neutralisation pathway and cannot be transported safely within the cell.

Hydroxyl radicals are typically formed through Fenton chemistry, in which hydrogen peroxide reacts with ferrous iron (Fe²⁺) to produce •OH and a hydroxide ion. The reaction is as follows:
Fe²⁺ + H₂O₂ → Fe³⁺ + •OH + OH⁻

Hydrogen peroxide is continually formed in the mitochondria as superoxide is dismutated, and iron is essential in many biological processes. However, when iron becomes unbound or loosely chelated, especially in the presence of inflammation or oxidative stress, it becomes available to drive Fenton reactions. This creates a high-risk situation where radical formation becomes uncontrolled.

These reactions most often occur in locations where both iron and hydrogen peroxide are present in close proximity, such as the mitochondria, nucleus, and lysosomes. Hydroxyl radicals are also generated in greater quantities during iron overload, chronic inflammation, reperfusion after ischemia, and ionising radiation exposure. Once formed, hydroxyl radicals cause direct strand breaks in DNA, peroxidise polyunsaturated fatty acids in membranes, and cause irreversible structural damage to proteins. Their indiscriminate nature makes them a major driver of tissue degeneration, neurological decline, and ageing-associated cellular dysfunction.

Peroxynitrite: Where Nitric Oxide and Superoxide Collide

Peroxynitrite (ONOO⁻) is not a free radical itself, but it is formed when two radicals, nitric oxide and superoxide, react with each other at nearly diffusion-limited rates. The reaction occurs so rapidly that it outcompetes the protective actions of superoxide dismutase, especially when both molecules are elevated. This often happens in inflamed, hypoxic, or metabolically overactive tissues.

Once formed, peroxynitrite is highly reactive and able to diffuse across membranes. One of its most damaging actions is the nitration of tyrosine residues in proteins, forming 3-nitrotyrosine, a modification that can alter protein folding, inhibit enzymatic activity, block signalling pathways, and mark proteins for degradation. This nitration disrupts critical functions such as mitochondrial respiration, cytoskeletal integrity, and receptor activity.

The accumulation of nitrated proteins has been documented in multiple degenerative diseases, including Parkinson’s, Alzheimer’s, atherosclerosis, and chronic infections. Because peroxynitrite arises from the collision of two signalling molecules, superoxide and nitric oxide, its formation represents a point of convergence between oxidative and nitrosative stress. In tissues where this convergence becomes frequent or sustained, such as the vascular endothelium, brain, heart, and immune system, the resulting damage accelerates dysfunction and ageing. Managing the levels of both superoxide and nitric oxide is thus essential not only for preserving cellular integrity but for preventing the misfiring of signalling networks that contribute to disease.

Ageing and the Escalation of Radical Formation

Age-related increases in ROS arise due to several overlapping factors:

1. Decline in mitochondrial efficiency: Damaged respiratory complexes elevate electron leakage (Squier, 2001).
2. Reduction in endogenous antioxidants: Enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) decrease with age (Sohal and Orr, 2012).
3. Impaired autophagy and mitophagy: Dysfunctional mitochondria accumulate, further promoting ROS generation (López-Otín et al., 2013).
4. Chronic inflammation (inflammaging): Persistent NF-κB activation drives ROS production via NADPH oxidase systems (Franceschi et al., 2007).

This oxidative shift destabilises DNA, proteins, lipids, and membranes—culminating in cell senescence, apoptosis, or oncogenic transformation.

ROS and Cancer: Mechanistic Links

The genotoxic potential of ROS is well established. Oxidative lesions like 8-oxoguanine mispair with adenine during DNA replication, generating point mutations (Loft and Poulsen, 1996). Peroxynitrite and hydroxyl radicals induce double-strand breaks and base oxidation, promoting genetic instability and carcinogenesis (Valko et al., 2006).

Additionally, ROS modulate cell signalling pathways, such as MAPK, PI3K/AKT, and p53, which regulate cell proliferation, survival, and DNA repair. Dysregulation of these redox-sensitive networks further predisposes aged tissues to malignant transformation.

Antioxidants: The Architecture of Redox Defence

Antioxidant systems act to neutralise free radicals before they initiate chain reactions. They operate via:

• Direct scavenging (e.g., ascorbate, glutathione)
• Metal chelation (e.g., flavonoids binding iron/copper)
• Enzymatic detoxification (e.g., SOD: O₂•⁻ → H₂O₂, Catalase: H₂O₂ → H₂O + O₂)

Vitamin C (ascorbate) functions as a water-soluble electron donor, directly quenching superoxide, peroxyl, and hydroxyl radicals, and regenerating oxidised vitamin E (Carr and Frei, 1999).

Vitamin E, a lipophilic chain-breaking antioxidant, prevents lipid peroxidation in polyunsaturated membranes. Polyphenols, abundant in plants, modulate redox signalling and inhibit pro-oxidant enzymes like xanthine oxidase.

Why Plants Have More Antioxidants Than Meat

Plants, being sessile, face constant environmental stress—UV, pathogens, oxidative metals. As a defence, they evolved robust secondary metabolite systems, such as flavonoids, carotenoids, and phenolic acids. These compounds protect their own cells and, when ingested, confer cross-species protective effects (Scalbert and Williamson, 2000).

Animals, by contrast, rely more heavily on endogenous enzymatic systems and behavioural avoidance. Muscle meat contains endogenous antioxidants such as carnosine, taurine, and CoQ10, but these are typically less diverse and lower in concentration than plant-derived compounds.

Radical Formation and Lifestyle: Alcohol, Exercise, Sleep, and Stress

• Alcohol metabolism (via CYP2E1) generates ROS and acetaldehyde, a DNA-damaging agent (Setshedi et al., 2010).
• Exercise induces transient oxidative stress but promotes long-term antioxidant upregulation—a hormetic adaptation (Gomez-Cabrera et al., 2008).
• Chronic stress elevates cortisol and adrenaline, both of which increase NADPH oxidase activity and mitochondrial ROS production (Chennaoui et al., 2015).
• Sleep deprivation suppresses melatonin, a potent mitochondrial antioxidant, and raises systemic oxidative load.

Fatty Acid Composition and Susceptibility to Lipid Peroxidation

• Polyunsaturated fatty acids (PUFAs) contain multiple double bonds, making them highly vulnerable to radical attack and lipid peroxidation. This generates toxic aldehydes (e.g., malondialdehyde) that form adducts with DNA and proteins (Ayala et al., 2014).
• Saturated fats, while chemically stable, may indirectly raise ROS through lipotoxicity and endoplasmic reticulum stress (Listenberger et al., 2003).

Balance, rather than exclusion, is critical. For example, omega-3 PUFAs are anti-inflammatory despite being oxidation-prone.

In Vivo Nitrite-Ascorbate Chemistry: Beyond Curing

In the human stomach, salivary nitrite—converted from dietary nitrate—meets acidic gastric juice and ascorbate, triggering the non-enzymatic reduction of nitrite to nitric oxide (Lundberg et al., 2008). This process supports:

• Antimicrobial activity in the upper GI tract
• Gastric mucosal blood flow via NO-mediated vasodilation
• Suppression of N-nitrosamine formation (Mirvish, 1995)

Without vitamin C, nitrite may form carcinogenic N-nitroso compounds through amine nitrosation. The co-presence of antioxidants is therefore not coincidental but a critical physiological strategy.

Nitrite and the Selective Chemistry of Antioxidants

Vitamin C uniquely reduces nitrite at low pH. Other antioxidants, like vitamin E or flavonoids, may inhibit nitrosation indirectly but do not catalyse NO formation. Nitrite–ascorbate interactions, therefore, represent a specialised chemical axis with implications for cardiovascular health, microbial defence, and dietary risk mitigation.

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Conclusion

Ageing is not merely the passage of time but the progressive failure of redox homeostasis. Mitochondria, designed to extract energy with surgical precision, begin to leak electrons—transforming oxygen into a saboteur. Free radicals emerge as both by-products and agents of decay, their burden rising with stress, poor sleep, poor diet, and environmental exposures.

Nitric oxide, sitting at the confluence of energy metabolism, vascular regulation, and immune defence, is both protector and precursor to oxidative damage—depending on context. Nutrients such as vitamin C and polyphenols, sourced from a diet rooted in plant–animal synergy, buffer these transitions.

The presence of antioxidants in plants and endogenous molecules in meat reflects a co-evolved nutritional alliance. Recognising and preserving this alliance, through food, sleep, movement, and biochemical insight, remains our best strategy against the cellular entropy we call ageing.

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