The tricarboxylic acid (TCA) cycle — also known as the Krebs cycle after Hans Krebs, or the citric acid cycle — is the central metabolic hub of aerobic life, a series of eight enzymatic reactions occurring in the mitochondrial matrix that oxidises acetyl-CoA derived from carbohydrates, fats, and proteins to CO2, generating the reduced electron carriers NADH and FADH2 that feed the electron transport chain for ATP production via Oxidative Phosphorylation. Far from being a simple energy-extraction pathway, the TCA cycle is the metabolic crossroads where catabolic and anabolic pathways converge: its intermediates serve as precursors for fatty acid synthesis, Amino Acids biosynthesis, Gluconeogenesis, haem production, and nucleotide synthesis. Every cell with functional mitochondria depends on the TCA cycle, making it one of the most ancient and conserved metabolic pathways in biology.
In clinical psychoneuroimmunology, the TCA cycle has acquired a significance that goes far beyond its textbook role as an energy pathway. The discovery that the TCA cycle is deliberately "broken" in activated M1 macrophages — with specific metabolites accumulating as signaling molecules rather than being processed for energy — has fundamentally reshaped our understanding of how inflammation, metabolism, and immune function are interconnected. Succinate accumulation stabilises HIF-1 to drive IL-1β production; Citrate accumulation provides substrate for pro-inflammatory lipid mediator synthesis and itaconate production; α-ketoglutarate promotes anti-inflammatory macrophage programming. The TCA cycle is therefore not merely a metabolic pathway but an immunoregulatory circuit, and its disruption is central to the metabolic basis of chronic low-grade inflammation, Macrophage Polarization, and the Warburg Effect that characterises activated immune cells.
Understanding the TCA cycle is essential for any cPNI practitioner because it provides the mechanistic link between nutritional status, metabolic flexibility, and immune function. Deficiencies in the cofactors required for TCA enzymes — including B vitamins (B1, B2, B3, B5), iron, Magnesium, and Manganese — directly impair energy production and can shift immune cells toward inflammatory metabolic programmes. Conversely, interventions that restore TCA cycle flux — such as correcting micronutrient deficiencies, supporting Metabolic flexibility through Intermittent fasting and physical activity, or providing anaplerotic substrates like Glutamine — can reshape immune cell metabolism from inflammatory to resolving phenotypes.
Per turn yield: 3 NADH + 1 FADH2 + 1 GTP + 2 CO₂ → ~10 ATP equivalents via Oxidative Phosphorylation
The TCA cycle begins when acetyl-CoA — a two-carbon acetyl group linked to coenzyme A — enters the cycle by condensing with the four-carbon molecule oxaloacetate (OAA). This reaction is catalysed by citrate synthase, producing the six-carbon tricarboxylic acid Citrate and releasing free CoA. Citrate synthase is the pace-setting enzyme of the cycle, inhibited allosterically by ATP, NADH, and citrate itself, ensuring that cycle flux matches cellular energy demand. This first step is essentially irreversible and commits the carbon skeleton to the cycle.
Citrate is then isomerised to isocitrate by the enzyme aconitase (aconitate hydratase), which first dehydrates citrate to cis-aconitate and then rehydrates it to isocitrate. Aconitase contains an iron-sulphur [4Fe-4S] cluster that is exquisitely sensitive to Oxidative Stress — reactive oxygen species and reactive nitrogen species can inactivate aconitase by disrupting this cluster, causing citrate to accumulate. This sensitivity makes aconitase a metabolic sensor of redox state and is one reason why Oxidative Stress shifts cells away from efficient TCA cycling. Importantly, cis-aconitate — the transient intermediate of this reaction — is the precursor for itaconate production via the enzyme aconitate decarboxylase 1 (ACOD1/IRG1), a reaction of enormous immunological significance discussed below.
The next step is the first of two oxidative decarboxylations. Isocitrate dehydrogenase (IDH) oxidises isocitrate to α-ketoglutarate (2-oxoglutarate), releasing one molecule of CO2 and reducing one molecule of NAD+ to NADH. This is a major regulatory point: IDH is activated by ADP and Ca2+ (signals of energy demand) and inhibited by ATP and NADH (signals of energy sufficiency). In Cancer, mutations in IDH1/2 produce the oncometabolite 2-hydroxyglutarate instead of α-ketoglutarate, which inhibits epigenetic demethylases and promotes tumorigenesis — a direct example of how TCA cycle dysfunction drives disease through metabolite-mediated epigenetic reprogramming.
α-Ketoglutarate then undergoes the second oxidative decarboxylation, catalysed by the α-ketoglutarate dehydrogenase complex (also called 2-oxoglutarate dehydrogenase). This multi-enzyme complex is structurally and mechanistically analogous to the pyruvate dehydrogenase complex and requires the same five cofactors: thiamine pyrophosphate (from Vitamin B1), lipoic acid, CoA (from Vitamin B5), FAD (from Vitamin B2), and NAD+ (from Vitamin B3). The reaction produces succinyl-CoA, releases CO2, and generates a second molecule of NADH. This enzyme is rate-limiting under many conditions and is inhibited by succinyl-CoA and NADH, providing feedback control. The dependence on five B-vitamin-derived cofactors underscores why B vitamins deficiency impairs TCA cycle flux and energy production.
Succinyl-CoA synthetase (also called succinate thiokinase) then cleaves the high-energy thioester bond in succinyl-CoA, producing Succinate and coupling this energy to the phosphorylation of GDP to GTP (which is energetically equivalent to ATP). This is the only step in the TCA cycle that produces a high-energy phosphate directly (substrate-level phosphorylation), analogous to the ATP-generating steps in glycolysis.
Succinate is oxidised to fumarate by succinate dehydrogenase (SDH), which is unique among TCA cycle enzymes because it is embedded in the inner mitochondrial membrane as Complex II of the electron transport chain. This dual role means SDH simultaneously participates in the TCA cycle and in Oxidative Phosphorylation, directly transferring electrons from succinate to ubiquinone (coenzyme Q10) via its FAD prosthetic group, generating FADH2 in the process. Because FADH2 enters the electron transport chain at Complex II rather than Complex I (where NADH enters), it generates approximately 1.5 ATP rather than the 2.5 ATP produced per NADH. SDH/Complex II is a critical node in Immunometabolism: in activated M1 macrophages, increased SDH activity and reverse electron transport from succinate drive mitochondrial reactive oxygen species (mROS) production, which further stabilises HIF-1 and amplifies IL-1β production.
Fumarase (fumarate hydratase) hydrates fumarate to malate in a freely reversible reaction. Finally, malate dehydrogenase oxidises malate back to oxaloacetate, reducing a third molecule of NAD+ to NADH. This reaction is thermodynamically unfavourable under standard conditions (positive delta G) but is pulled forward by the highly favourable citrate synthase reaction that immediately consumes oxaloacetate, keeping its concentration extremely low and driving the equilibrium.
In total, one turn of the TCA cycle produces: 3 NADH (at isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase), 1 FADH2 (at succinate dehydrogenase/Complex II), 1 GTP (at succinyl-CoA synthetase), and 2 CO2 (at the two decarboxylation steps). The three NADH and one FADH2 feed into the electron transport chain, where they drive proton pumping across the inner mitochondrial membrane and ultimately generate approximately 9-10 ATP per turn of the cycle via Oxidative Phosphorylation. Since one molecule of Glucose produces two molecules of acetyl-CoA (via pyruvate dehydrogenase), the TCA cycle turns twice per glucose, contributing approximately 20 ATP of the total ~30-32 ATP generated from complete glucose oxidation.
Perhaps the most important insight from Immunometabolism for cPNI practice is that the TCA cycle is deliberately fragmented in activated M1 macrophages, and the accumulating intermediates serve as potent immunoregulatory signals. When macrophages are activated by LPS or IFN-γ, the TCA cycle "breaks" at two critical points, creating what has been called the "broken Krebs cycle" — a metabolic rewiring that prioritises immune defence over energy production.
The first break occurs after citrate. Activated macrophages massively upregulate the mitochondrial citrate carrier (CIC/SLC25A1), which exports Citrate from the mitochondrial matrix to the cytoplasm. There, ATP citrate lyase cleaves citrate back into acetyl-CoA and oxaloacetate. The cytoplasmic acetyl-CoA serves multiple pro-inflammatory functions: it provides substrate for fatty acid synthesis (required for membrane biosynthesis during macrophage activation and for production of pro-inflammatory Prostaglandins and leukotrienes from Arachidonic acid), and it serves as the acetyl donor for histone acetylation, opening chromatin at inflammatory gene loci and amplifying transcription of Cytokines like TNF-α and IL-6. This epigenetic dimension — acetyl-CoA from the broken TCA cycle driving histone acetylation at inflammatory loci — is a profound example of how metabolism directly controls gene expression in immune cells.
Simultaneously, the enzyme ACOD1 (also known as IRG1, immune-responsive gene 1) is massively upregulated in activated macrophages. ACOD1 diverts cis-aconitate — the intermediate between citrate and isocitrate — to produce itaconate, a metabolite that does not exist in the classical TCA cycle but is one of the most abundant metabolites in activated macrophages. Itaconate functions as an endogenous anti-inflammatory brake: it inhibits SDH (Complex II), reducing succinate oxidation and the associated inflammatory mROS production; it activates Nrf2, the master transcription factor for antioxidant defence; it inhibits glycolysis by targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH); and it modifies proteins through a post-translational modification called itaconation, including modification of GAPDH and the inflammasome adaptor protein NLRP3. Itaconate therefore represents an elegant built-in resolution mechanism: the same metabolic rewiring that produces inflammatory signals (citrate export, succinate accumulation) simultaneously generates its own anti-inflammatory counterpart.
The second break occurs at Succinate. Multiple mechanisms drive succinate accumulation in activated macrophages: increased glutamine-dependent anaplerosis feeds carbon into the cycle via α-ketoglutarate, but the itaconate-mediated inhibition of SDH prevents succinate from being oxidised to fumarate. Additionally, the "aspartate-argininosuccinate shunt" — where aspartate and citrulline from the urea cycle feed into the TCA via fumarate — and the GABA shunt both contribute succinate. The accumulated Succinate has two major pro-inflammatory effects: (1) it inhibits prolyl hydroxylase domain (PHD) enzymes by competing with α-ketoglutarate, thereby stabilising HIF-1α even under normoxic conditions ("pseudohypoxia"), which drives transcription of IL-1β and glycolytic enzymes like GLUT1; and (2) when oxidised by SDH, it drives reverse electron transport at Complex I, generating mitochondrial ROS that further stabilise HIF-1 and activate the NLRP3 inflammasome. Succinate can also be released extracellularly, where it acts as a danger signal via the SUCNR1 (GPR91) receptor on neighbouring cells, amplifying local inflammation.
In contrast to the pro-inflammatory roles of citrate and succinate, α-ketoglutarate (2-oxoglutarate) acts as an anti-inflammatory metabolite. A high α-ketoglutarate-to-succinate ratio promotes M2 macrophage polarization through several mechanisms: α-ketoglutarate is a required co-substrate for the PHD enzymes that degrade HIF-1α (opposing the succinate-driven HIF stabilisation); it serves as co-substrate for Jumonji domain histone demethylases (JMJD3) that activate anti-inflammatory gene programmes; and it promotes fatty acid oxidation over glycolysis. Glutamine — the most abundant amino acid in plasma — is a major source of α-ketoglutarate via glutaminolysis, which is why Glutamine supplementation has been proposed as an anti-inflammatory intervention in cPNI. The α-ketoglutarate/succinate ratio can therefore be viewed as a metabolic rheostat determining the inflammatory state of macrophages.
Because TCA cycle intermediates are continuously siphoned off for biosynthetic purposes (cataplerosis), they must be replenished through anaplerotic ("filling up") reactions to maintain cycle flux. The most important anaplerotic reactions include: pyruvate carboxylase converting pyruvate to oxaloacetate (requiring biotin/Vitamin B7); glutaminase converting Glutamine to glutamate, then to α-ketoglutarate; branched-chain amino acid (BCAAs) metabolism feeding into succinyl-CoA; and aspartate/asparagine transamination to oxaloacetate. In immune cells, glutamine-dependent anaplerosis is quantitatively the most important, which is why Glutamine is considered a conditionally essential amino acid during inflammation and critical illness — demand outstrips supply.
Cataplerotic reactions — the withdrawal of intermediates for biosynthesis — include citrate export for fatty acid synthesis and histone acetylation, oxaloacetate withdrawal for Gluconeogenesis (in liver) and aspartate synthesis, succinyl-CoA withdrawal for haem biosynthesis, and α-ketoglutarate withdrawal for amino acid transamination and collagen hydroxylation (via prolyl and lysyl hydroxylases that require α-ketoglutarate, iron, and vitamin C). The balance between anaplerosis and cataplerosis determines whether the TCA cycle can maintain flux or becomes depleted — a consideration directly relevant to nutritional interventions in cPNI.
The TCA cycle interfaces with virtually every major metabolic pathway. Glycolysis feeds pyruvate into the cycle via acetyl-CoA (pyruvate dehydrogenase complex), or directly via anaplerotic carboxylation to oxaloacetate. Beta-oxidation feeds the cycle with acetyl-CoA from fatty acid oxidation — each turn of the beta-oxidation spiral generates one acetyl-CoA that enters the TCA cycle. Amino Acids catabolism feeds into the cycle at multiple points: glutamate/glutamine at α-ketoglutarate, aspartate at oxaloacetate, valine/isoleucine at succinyl-CoA, phenylalanine/tyrosine at fumarate. Gluconeogenesis withdraws oxaloacetate (via phosphoenolpyruvate carboxykinase) to generate glucose — this is why fatty acids cannot be converted to glucose in mammals (acetyl-CoA enters as a two-carbon unit but both carbons are lost as CO2 before oxaloacetate is regenerated). Oxidative Phosphorylation receives the NADH and FADH2 generated by the cycle to produce the bulk of cellular ATP. The TCA cycle is truly the metabolic hub where fuel substrates, biosynthetic needs, and energy production converge.