Isotopic labeling of metabolic water with ¹⁸ O ₂

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Rationale Water is the medium of life, is involved in biochemical reactions, and is exchanged among internal pools and with the water in the external environment of organisms. Understanding these processes can be improved by isotopically labeling the metabolic water that is produced inside the cells of organisms during aerobic respiration. Methods Here we describe a new method for isotopically labeling cellular water by incubating microbes and plant tissues in air enriched in 18 O 2 . As oxygen gas is reduced during respiration, H 2 18 O is produced. The rate of H 2 18 O production and the synthesis of biomolecules that incorporate 18 O from H 2 18 O can be quantified using cavity ringdown spectrometry and isotope ratio mass spectrometry. Results For Escherichia coli in solution culture, soil microbial communities, and respiring tissues of plants, the amount of H 2 18 O produced was strongly correlated with that of 18 O 2 consumed during incubations. Measurements of 18 O in DNA, microbial biomass, and CO 2 showed that metabolic water was an important substrate in biosynthesis reactions. Conclusions Any organism with aerobic respiration is amenable to labeling with 18 O 2 , and the method described here enables a new approach to investigate questions regarding plant and microbial physiology. In plants, 18 O introduced as metabolic water could be tracked as it moves between living cells and exchanges with external water. For probing soil microbial physiology, the method described here has the advantage over the application of exogenous H 2 18 O of not increasing the soil moisture, a disturbance that can affect microbial metabolism.