Isotopic labeling of metabolic water with ¹⁸ O₂

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.

Here we describe a new method for isotopically labeling cellular water by incubating microbes and plant tissues in air enriched in ¹⁸ O₂ . As oxygen gas is reduced during respiration, H₂ ¹⁸ O is produced. The rate of H₂ ¹⁸ O production and the synthesis of biomolecules that incorporate ¹⁸ O from H₂ ¹⁸ O can be quantified using cavity ringdown spectrometry and isotope ratio mass spectrometry.

For Escherichia coli in solution culture, soil microbial communities, and respiring tissues of plants, the amount of H₂ ¹⁸ O produced was strongly correlated with that of ¹⁸ O₂ consumed during incubations. Measurements of ¹⁸ O in DNA, microbial biomass, and CO₂ showed that metabolic water was an important substrate in biosynthesis reactions.

Any organism with aerobic respiration is amenable to labeling with ¹⁸ O₂ , 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 H₂ ¹⁸ O of not increasing the soil moisture, a disturbance that can affect microbial metabolism.