The Hidden Cause of Ketosis: CO₂ Holdup
Two Forces Governing Cellular Homeostasis
Cellular homeostasis is regulated by two fundamental forces: the proton motive force (PMF) (Abad, 2011) and pH (Occhipinti & Boron, 2019). In the ruminal epithelium, homeostasis is largely driven by bicarbonate (HCO₃⁻) transport and high intracellular HCO₃⁻ concentrations (Bilk et al., 2005; Lee & Hong, 2020). This creates a striking contrast: the ruminal fluid is rich in dissolved CO₂ (dCO₂), while intracellular CO₂ remains low due to rapid hydration to HCO₃⁻.
Aquaporins transport both water and CO₂ into cells (Endeward et al., 2017; Zhong et al., 2020), where intracellular carbonic anhydrase (CA) quickly converts them into HCO₃⁻ and protons (Rabbani et al., 2021; Vilas et al., 2015). This maintains the HCO₃⁻ gradient. Thus, the CO₂/HCO₃⁻ gradient better explains epithelial homeostasis than pH, since the pH scale is a quotient and does not measure absolute concentrations.
High ruminal CO₂ favors HCO₃⁻ dehydration (rumen side), while high intracellular HCO₃⁻ favors CO₂ hydration (cell side). Cells evolving in CO₂- and HCO₃⁻-rich environments required PMF to stabilize homeostasis. For example, red blood cells accumulate Cl⁻ to increase HCO₃⁻ carrying capacity (Klocke, 1987), and bacteria use futile cycles to survive under extreme conditions (Buurman et al., 1991). This may explain the apparent coupling of pH and PMF.
This is relevant because catabolism enhances HCO₃⁻ generation to replenish intracellular pools, while high HCO₃⁻ promotes anabolism. Thus, intracellular HCO₃⁻ balance profoundly affects energy metabolism and cellular function (Alka & Casey, 2014; Blombach & Takors, 2015). Since the ruminal epithelium depends on CO₂ and H₂O absorption via aquaporins for HCO₃⁻ formation (Rackwitz & Gäbel, 2018), reduced CO₂/H₂O uptake — as in dehydration — may trigger catabolism through cellular dehydration.
Ketosis: A Unifying Theory
Ketosis is a major metabolic disorder in dairy cattle with significant economic consequences. Traditionally, primary ketosis (post-calving) and secondary ketosis (starvation-induced) have been viewed as separate entities (Baird et al., 1972; Oetzel, 2007). However, they may share a common mechanism: cellular dehydration and HCO₃⁻ depletion.
Secondary ketosis: As early as 1960, Krebs described the effect of starvation on hepatic ketogenesis. Later studies suggested that intracellular acidification may trigger this response.
Primary ketosis: Although multiple theories exist, it is established that AMPK activation — the cellular energy sensor — induces ketogenesis, even when energy supply is adequate.
Both conditions may converge on the same pathway: CO₂ holdup or starvation → reduced dCO₂/H₂O uptake → intracellular HCO₃⁻ depletion → cellular dehydration → catabolic activation → ketogenesis.
Cellular Dehydration as the Common Trigger
Under normal conditions, the ruminal epithelium requires the joint absorption of CO₂ and H₂O via aquaporins. Intracellularly, CA combines these molecules into HCO₃⁻ and H⁺, which are then used for Na⁺ and SCFA absorption (Rackwitz & Gäbel, 2018). Up to 80% of CO₂ turnover in the rumen epithelium occurs through this “aquaporin cycling” (Veenhuizen et al., 1988). Indeed, dCO₂ enhances SCFA absorption (Ash & Dobson, 1963).
During starvation, SCFA and dCO₂ production decline, limiting intracellular HCO₃⁻/H₃O⁺ formation and leading to cellular dehydration.
After calving, high-production diets may trigger CO₂ holdup: physicochemical changes prevent CO₂ effervescence, causing liquid dCO₂ accumulation. This contributes to ruminal acidosis (Laporte-Uribe, 2023), ruminal hyperosmolarity (Ash & Dobson, 1963), and reduced H₂O absorption (Dobson, 1971; Lodemann & Martens, 2006).
In both cases, ketosis (ketone body formation from fat mobilization; White, 2015) can be seen as a compensatory mechanism to restore intracellular CO₂/H₂O pools for HCO₃⁻ and H⁺ generation.
Consequences of Cellular Dehydration
Reduced HCO₃⁻ formation: Limited CO₂/H₂O uptake reduces intracellular HCO₃⁻, leading to acidification.
Impaired SCFA and Na⁺ absorption: Declining HCO₃⁻ impairs nutrient uptake, resulting in SCFA accumulation.
AMPK activation: Reduced HCO₃⁻ and nutrient uptake activate AMPK, driving fat mobilization and ketogenesis as an alternative energy source.
Thus, both primary and secondary ketosis may result from the same fundamental disturbance: cellular dehydration due to impaired CO₂/H₂O absorption and disrupted aquaporin cycling.
Continuous dCO₂ Monitoring as Prevention
This theory underscores the importance of continuous dCO₂ monitoring. By detecting early signs of CO₂ holdup (diet-induced) or reduced fermentative activity (starvation), dietary interventions can be implemented proactively to minimize cellular dehydration and prevent ketogenesis.
