By Eben van Tonder, 4 August 2025
Introduction
This article brings together an advanced exploration of nitric oxide (NO), superoxide (O₂⁻), peroxynitrite (ONOO⁻), and iron oxidation to provide a unified understanding of two seemingly unrelated systems: human asthma and cured meat colour chemistry. The discussion explores how biological and postmortem processes converge on a small set of redox reactions that shape both health and food technology.
Section 1: Myoglobin, Iron States, and Meat Colour
Iron in Myoglobin and Oxygen Binding
Myoglobin contains a heme prosthetic group, in which an iron atom sits at the centre of a porphyrin ring. This iron can switch between two oxidation states: the ferrous state (Fe²⁺), which is capable of reversibly binding oxygen or nitric oxide, and the ferric state (Fe³⁺), which is oxidised and unable to bind oxygen, leading to brown discolouration (metmyoglobin). Oxygen binds to Fe²⁺ in a bent geometry and is stabilised by a distal histidine. When oxygen is released, such as during muscular activity, the iron usually remains in the ferrous state unless oxidised by reactive oxygen species (ROS).
Importantly, the binding of oxygen to Fe²⁺ does not involve a direct transfer of electrons to oxygen; the bond formed is primarily coordinate. Nevertheless, prolonged exposure to oxygen or oxidative conditions can oxidise Fe²⁺ to Fe³⁺. Nitric oxide can also bind to Fe²⁺ similarly to oxygen but forms a more stable complex. If NO binds to Fe³⁺, it still induces the red cured colour associated with nitrosylmetmyoglobin, although this bond is generally weaker and less stable.
Why and When Myoglobin Releases Oxygen
Oxygen is released from myoglobin when the partial pressure of oxygen in the surrounding tissue is low, making oxygen release thermodynamically favourable. Other factors include the presence of competitive ligands such as NO and changes in muscle environment, such as pH and ionic strength, which can alter the conformation of the heme pocket and weaken the bond to oxygen. Once oxygen is released, the exposed Fe²⁺ is vulnerable to oxidation, particularly in the presence of superoxide or hydrogen peroxide.
Pigment States in Meat
The colour of meat is determined by the state of the iron in myoglobin and the ligand attached to it. Deoxymyoglobin, in which Fe²⁺ is unbound to any ligand, is purple-red. Oxymyoglobin, where Fe²⁺ is bound to oxygen, is bright red. Metmyoglobin, where Fe is in the Fe³⁺ state and unbound, is brown. When NO binds to Fe²⁺, it forms nitrosylmyoglobin, which is bright pink. Even when NO binds to Fe³⁺, the resulting nitrosylmetmyoglobin still gives a red-pink hue. Cooking denatures the myoglobin protein, and when NO is bound during this process, it locks in the colour, forming the stable nitrosylhemochrome pigment responsible for the pink colour of cured meat.
Section 2: NO, Superoxide, and ONOO⁻ Chemistry
Core Reaction
Peroxynitrite is formed when nitric oxide reacts with superoxide in a rapid, diffusion-limited reaction: NO + O₂⁻ → ONOO⁻
Pathways for NO
Nitric oxide reacts differently depending on the oxidative environment. In the presence of abundant oxygen, NO combines with O₂ to form nitrogen dioxide (NO₂), which can further react to form nitrate (NO₃⁻). When superoxide is present, NO reacts to form peroxynitrite. In hypoxic or low-oxygen conditions, nitrite (NO₂⁻) can be enzymatically or non-enzymatically reduced back to NO. This reaction is enhanced by reducing agents like ascorbate or proteins such as deoxyhemoglobin.
Pathways After ONOO⁻ Formation
From bronchodilation to bacon, redox control defines functionality. Iron in the Fe²⁺ state enables life, colour, and oxygen transport. Once oxidised to Fe³⁺, it becomes dysfunctional and discoloured. Nitric oxide serves as a messenger and protector, but becomes hazardous when oxidative stress is high and superoxide is unopposed. This unified framework allows us to understand asthma therapies, meat curing, and oxidative pathology through a single lens: redox regulation. Strategic use of antioxidants, dietary nitrates, and enzymatic defences provides both practical applications and biological insight.
Peroxynitrite is highly reactive and can nitrate tyrosine residues in proteins, oxidise Fe²⁺ to Fe³⁺, and cause oxidative damage to lipids, DNA, and mitochondrial enzymes. These reactions impair cellular signalling, energy production, and contribute to inflammation and tissue injury.
Section 3: Antioxidants and Defence Mechanisms
In Tissue and Meat
Superoxide dismutase (SOD) catalyses the dismutation of superoxide into hydrogen peroxide and oxygen, a critical defence step. Catalase and glutathione peroxidase further break down hydrogen peroxide into water and oxygen, preventing the formation of hydroxyl radicals via the Fenton reaction. The Fenton reaction, involving Fe²⁺ and H₂O₂, produces the highly reactive hydroxyl radical (•OH). Antioxidants like vitamin C serve multiple protective functions: they scavenge ROS, reduce Fe³⁺ back to Fe²⁺, and facilitate the conversion of nitrite to NO, especially in curing environments.
Section 4: Asthma and ONOO⁻
NO and Superoxide in Asthma
Asthma is marked by chronic inflammation in the airways. This upregulates inducible nitric oxide synthase (iNOS), leading to elevated NO production. At the same time, immune cells like neutrophils and macrophages generate superoxide. The convergence of NO and superoxide promotes the formation of peroxynitrite, which damages epithelial cells, remodels airway tissue, and triggers bronchoconstriction.
Role of Asthma Medications
Medications like Symbicort, a combination of budesonide and formoterol, reduce inflammation and promote bronchodilation. Budesonide alone is a corticosteroid that downregulates inflammatory cytokines and iNOS expression. Asthavent (salbutamol) is a fast-acting β₂-agonist that opens airways. Holding one’s breath after inhalation increases medication deposition in the lower airways, potentially enhancing effectiveness.
Section 5: Beetroot, NO, and Dietary Strategy
Nitrate Pathway
Beetroot is rich in dietary nitrate (NO₃⁻), which is reduced in the body to nitrite (NO₂⁻) and then to NO, especially under hypoxic conditions. This pathway is beneficial in asthma and exercise, where oxygen availability is limited. Increased NO levels enhance vasodilation, oxygen delivery, and respiratory efficiency.
Risk of ONOO⁻
The formation of peroxynitrite is primarily a concern when superoxide levels are high and not neutralised. This imbalance can be mitigated by antioxidants like vitamin C, N-acetylcysteine (NAC), polyphenols, and omega-3 fatty acids, which lower oxidative stress and preserve NO signalling.
Section 6: Peroxynitrite Formation and Blockers
Formation Conditions
Peroxynitrite is formed under conditions of chronic inflammation, mitochondrial dysfunction, and persistent infection. Diseases like tuberculosis, asthma, and chronic sinusitis elevate both NO and superoxide production, increasing ONOO⁻ levels.
Blockers of ONOO⁻
Vitamin C directly scavenges superoxide, reduces Fe³⁺ to Fe²⁺, and converts nitrite to NO, preventing ONOO⁻ formation. NAC increases intracellular glutathione, enhancing cellular detoxification. Ascorbate also supports the cured meat pigment by protecting Fe²⁺. Enzymes like catalase and glutathione peroxidase degrade hydrogen peroxide, removing key substrates that fuel further oxidation.
Section 7: Postmortem Chemistry in Meat
How Fresh Meat Turns Brown
Fresh meat turns brown due to the oxidation of Fe²⁺ in myoglobin to Fe³⁺ when exposed to oxygen. This oxidised form, metmyoglobin, no longer binds oxygen and appears brown. If the meat is submerged in water, oxygen diffusion is reduced, slowing the oxidation process.
NO Protects Colour
Nitric oxide binds to Fe²⁺ in myoglobin, forming nitrosylmyoglobin, which is more resistant to oxidation. Even when NO binds to Fe³⁺, it forms nitrosylmetmyoglobin, which still gives a pinkish cured hue, although less stable than the Fe²⁺ version.
Cooking Locks Colour
During cooking, proteins unfold and the nitrosylmyoglobin is converted to nitrosylhemochrome, a stable pigment responsible for the pink colour of cooked cured meats. This colour remains stable up to temperatures of about 65–75°C. Beyond that, denaturation becomes dominant, potentially fading the pink hue.
Section 8: ATP, Electron Flow, and Oxygen’s Role
How Oxygen Pulls Electrons
In mitochondria, oxygen acts as the terminal electron acceptor in the electron transport chain (ETC). Electrons donated by NADH and FADH₂ pass through complexes I to IV, with each transfer releasing energy that pumps protons across the mitochondrial membrane. At complex IV (cytochrome c oxidase), O₂ receives four electrons and is reduced to two molecules of H₂O. This electron pull is essential for maintaining the gradient that powers ATP synthesis.
Disruption During Inflammation and Ischaemia
In conditions such as chronic inflammation, heart attacks, or strokes, oxygen supply becomes limited (hypoxia) or is interrupted (ischaemia). This disrupts electron flow, leading to electron leakage from complexes I and III, where they prematurely reduce oxygen to superoxide. Mitochondria become sites of oxidative stress, especially when NO is also elevated.
NO’s Dual Role in ATP Regulation
NO competes with oxygen at cytochrome c oxidase. At low concentrations, NO can reversibly bind, modulating respiration and protecting against ROS formation by temporarily blocking O₂ reduction. However, under sustained inflammatory or hypoxic stress, NO’s presence becomes insufficient to prevent superoxide formation. Superoxide reacts rapidly with NO to form ONOO⁻, compromising both NO signalling and ATP production. The presence of both NO and superoxide thus becomes highly problematic: what was once a protective molecule turns into a contributor to damage.
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Ascorbate and Peroxynitrite: Revisiting the Antioxidant That Bridges Curing and Physiology
How Peroxynitrite Forms and Harms the Body
Peroxynitrite (ONOO⁻) is a reactive nitrogen species formed when superoxide (O₂⁻·) combines with nitric oxide (·NO). The reaction is rapid and spontaneous:
O₂⁻· + ·NO → ONOO⁻
Although not itself a radical, ONOO⁻ is highly reactive and dangerous. Under acidic conditions (such as in inflamed tissue or mitochondria), ONOO⁻ becomes protonated to peroxynitrous acid (ONOOH). ONOOH is unstable and rapidly decomposes into:
Hydroxyl radicals (·OH)
Nitrogen dioxide radicals (·NO₂)
These are among the most damaging species in biology. They attack lipids, DNA, and proteins, leading to oxidative chain reactions, membrane damage, and nitrotyrosine formation—hallmarks of oxidative and nitrosative stress.
Vitamin C (ascorbate, in its anionic form as AscH⁻) directly neutralises ONOO⁻ before this breakdown occurs. The core mechanism is as follows:
ONOO⁻ + AscH⁻ → NO₂⁻ + Asc·⁻ + OH⁻
Here, ascorbate donates a single electron to ONOO⁻. This reaction:
Converts peroxynitrite to harmless nitrite (NO₂⁻)
Forms a stable ascorbyl radical (Asc·⁻)
And releases a hydroxide ion (OH⁻), slightly alkalinising the local environment
This means ascorbate intercepts ONOO⁻ upstream, preventing the generation of the ·OH and ·NO₂ radicals that would otherwise propagate damage. The ascorbyl radical is relatively non-reactive and can either be recycled back to ascorbate (by NADH or other reductants) or dimerise to form dehydroascorbic acid.
This direct one-electron reduction of ONOO⁻ makes vitamin C one of the few physiological molecules capable of stopping peroxynitrite in its tracks. Unlike other antioxidants that merely slow oxidative cascades, ascorbate prevents the reaction from ever starting.
Experimental Evidence: Kirsch & de Groot (2000)
This mechanism was confirmed in detail by Kirsch and de Groot (2000) in a series of cellular and biochemical experiments published in The Journal of Biological Chemistry. They observed that:
Ascorbate reacts rapidly with peroxynitrite, with a second-order rate constant of approximately 10⁵ M⁻¹·s⁻¹. This means that ascorbate competes successfully with biomolecules for ONOO⁻.
Tyrosine nitration was markedly inhibited in the presence of ascorbate, indicating that peroxynitrite was intercepted before decomposing into ·NO₂.
Oxidative stress-induced DNA damage and lipid peroxidation were significantly reduced in ascorbate-rich systems, both in vitro and in intact cell models.
Even when cellular glutathione was depleted, ascorbate alone provided significant protection, demonstrating its effectiveness as a standalone scavenger.
Their conclusion was unambiguous: ascorbate is “a potent antioxidant against peroxynitrite-induced oxidative damage, capable of intercepting peroxynitrite and protecting cells from its cytotoxic effects.”
This provides strong experimental confirmation of what modern nutritional and pharmacological science has increasingly recognised: vitamin C is not only a dietary antioxidant but a frontline defence against one of the most damaging reactive nitrogen species in human biology.
Vitamin C Supplements: A Clinical Consideration
Given this remarkable capacity to neutralise peroxynitrite, a natural question arises: as one ages, or faces chronic inflammation, is it necessary to supplement vitamin C, even as a simple sulphate tablet?
Our Needs for Vitamin C Increase with Age
Ageing is associated with several factors that increase oxidative and nitrosative stress:
Mitochondrial dysfunction and reduced efficiency in ROS scavenging
Chronic low-grade inflammation (inflammaging)
Declining dietary intake due to reduced appetite or absorption efficiency
These contribute to increased demand for vitamin C. Many researchers argue that the current recommended daily allowance (RDA) of 90 mg for men and 75 mg for women is too low for optimal cellular defence, particularly in high-stress or inflammatory states. For example, smokers, individuals with chronic illness, or people exposed to urban pollution all experience elevated turnover of vitamin C.
What Dosage, When, and How?
Most clinical protocols recommend 500 mg to 1,000 mg per day for therapeutic purposes. The tolerable upper intake level (UL) is set at 2,000 mg per day, beyond which gastrointestinal side effects such as diarrhoea may occur in some individuals.
Best taken:
– With meals to improve uptake and reduce gastric irritation
– In divided doses (for example, 500 mg twice a day) to maintain steady plasma levels
– Buffered forms like sodium ascorbate may be better tolerated by sensitive stomachs
– Liposomal vitamin C offers superior bioavailability and is often used in clinical settings
It Protect Against Stroke, Heart Attack, or Inflammatory Conditions
There is a growing body of evidence supporting vitamin C’s protective role in the following conditions:
Stroke and heart attack (ischemia-reperfusion injury):
Ascorbate reduces oxidative damage during the critical window when blood supply returns to previously starved tissue. It scavenges ONOO⁻ and supports endothelial NO signalling, potentially limiting infarct size.
Asthma:
Asthma is marked by elevated NO and ROS in the airways. Vitamin C has been shown to reduce bronchial hyperresponsiveness, especially during viral infections, heavy exercise, or exposure to irritants. Some studies show it improves lung function in subgroups of asthmatic patients.
Tuberculosis and chronic sinusitis:
These conditions involve persistent inflammation and immune activation. Vitamin C supports epithelial repair, collagen synthesis, and mitigates oxidative damage in chronically inflamed tissues.
Clot prevention and blood flow:
While not a blood thinner in the pharmaceutical sense, vitamin C improves endothelial function, preserves NO bioavailability, and reduces oxidative stress on vessel walls. These effects support better circulation and may reduce thrombotic risk indirectly.
Other Potential Benefits
Enhances iron absorption, especially non-heme iron from plant sources
Regenerates vitamin E, another key antioxidant
May slow cognitive decline via reduction in oxidative burden
Supports immune defence, particularly under stress or during infection
This comprehensive role, spanning molecular protection, immune modulation, and vascular health, places vitamin C at the intersection of meat science, respiratory physiology, and chronic disease management. It is not only the key to pink ham, but quite possibly, to preserving cellular integrity across the lifespan.
The Role of Ascorbate in Curing: Iron Reduction and Colour Stability
Beyond its physiological role, ascorbate is pivotal in the curing of meat precisely because of its interaction with iron in myoglobin. The red colour of cured meat results from the formation of nitrosylmyoglobin, a complex between nitric oxide (NO) and the iron atom in the haem group of myoglobin. While NO can bind to both Fe³⁺ (ferric) and Fe²⁺ (ferrous) iron, the binding to Fe²⁺ is essential for achieving the stable, pink cured colour associated with ham and bacon. The Fe³⁺-NO complex is much less stable and prone to discolouration or breakdown, particularly during heat treatment. Therefore, ascorbate is added to curing brines to chemically reduce Fe³⁺ back to Fe²⁺, ensuring that when NO is generated from nitrite, it binds to iron in its reduced state and forms a heat-stable, bright pink pigment.
From a dietary perspective, however, it is important to note that ascorbate added during curing does not persist through cooking. Whether in bacon fried in a pan or ham cooked in a water bath, ascorbate is largely consumed in the curing reactions and degraded by heat. Its antioxidant benefits, therefore, are technological rather than nutritional in this context. While some residual vitamin C may survive in mildly cooked cured products, particularly those with shorter heat exposure, the majority is lost during standard cooking processes. This means that cured meat is not a significant dietary source of vitamin C, even if ascorbate was essential to its production.
Conclusion: A Biochemical Bridge Between Meat and Medicine
At the heart of both asthma and meat curing lies the same elusive molecule: nitric oxide (NO). Its capacity to signal, stabilise, and defend is matched only by its fragility. Whether in inflamed lung tissue or a curing brine, NO faces the same biochemical threats, chief among them the oxidising power of superoxide and its rapid coupling with NO to form peroxynitrite (ONOO⁻). This toxic compound unites two seemingly unrelated fields: the pathology of chronic inflammation and the chemistry of meat discolouration.
Iron serves as the molecular hinge. In its reduced ferrous (Fe²⁺) state, it binds nitric oxide to form the characteristic pink hue of cured meat, or in physiology, facilitates oxygen storage and enzymatic signalling. But once oxidised to ferric (Fe³⁺) iron, its binding affinity changes, its colour fades, and its function falters. In both contexts, human tissue and meat muscle, the oxidation of iron is a loss of structure, signal, and meaning.
Ascorbate emerges as the guardian molecule. In curing, it reduces Fe³⁺ back to Fe²⁺, ensuring that NO binds efficiently and forms a stable nitrosyl complex, resistant to heat and oxidation. This explains why ascorbate dramatically improves colour yield and retention during thermal processing. In bacon, ham, and frankfurters, the Fe²⁺–NO complex resists denaturation under cooking conditions, whereas without ascorbate, discolouration and pigment breakdown are far more likely. Ascorbate not only accelerates the reduction of nitrite to NO, essential for the curing reaction, but also preserves iron in its most reactive, stable, and colour-yielding form.
In human biology, the same electron-donating power allows ascorbate to neutralise peroxynitrite directly, preventing its decomposition into the highly damaging hydroxyl and nitrogen dioxide radicals. By converting ONOO⁻ to benign nitrite, ascorbate shields the lungs, the vasculature, and the nervous system from nitrosative catastrophe. This mechanism is not theoretical. It has been experimentally confirmed in cellular models, where vitamin C dramatically reduces protein nitration, lipid peroxidation, and DNA fragmentation.
That a single molecule could perform such parallel functions, one in a curing room, the other in the alveoli of a struggling asthmatic, invites a deeper understanding of what meat science and medical biochemistry truly share. Nitric oxide, peroxynitrite, iron, and ascorbate form a tightly interconnected system. Their behaviour governs the colour of ham, the breath of a child, and the boundary between health and degeneration.
In exploring these shared mechanisms, we find not only a unified framework for curing and inflammation, but also a reminder: food chemistry and human physiology speak the same biochemical language. And in both, ascorbate speaks last.
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