Decoupling of CO2 and O2 fluxes reveals oxygen-independent decarboxylation in anoxic mammalian cells

Abstract

Canonical understanding of cellular respiration holds that CO2 production tracks oxygen consumption, and mammalian cells deprived of oxygen are thought to rely on lactate fermentation, which yields no net CO2. Because standard respirometry infers CO2 flux either from oxygen consumption or from extracellular acidification rather than measuring it directly, a CO2 flux decoupled from oxygen would go undetected or be misattributed to glycolysis. Using direct measurement of CO2 and O2 fluxes from the gas phase in anoxic mammalian cells, we show that a substantial CO2 efflux persists after oxygen is fully removed. This anoxic flux scales linearly with normoxic CO2 and with cell mass, and exhibits a lower activation energy than normoxic respiration, suggesting a distinct rate-limiting chemistry. Pyruvate and -ketobutyrate increased the anoxic CO2 flux while lactate dehydrogenase inhibition abolished the pyruvate-induced increase, supporting a role for cytosolic NAD+ regeneration in oxygen-independent decarboxylation. 13C tracing identified a substantial glutamine-derived component, consistent with mitochondrial decarboxylation, and a smaller glucose-derived component associated with the cytosolic pentose phosphate pathway. Direct CO2 measurement thus reframes lactate dehydrogenase as an enabler of oxygen-independent decarboxylation.