Fig. 2. Data showing validation of method parameters. Each data point represents measurement of helium emitted from a single animal in one trial after completing the experimental protocol (i.e. an entire sequence of heliox exposure, nitrox flushing of chamber, and sealed chamber equilibration; helium assessed by gas chromatography of an air sample from the chamber). Different symbols indicate different animals. (A) Helium yield from animals varied with the duration of pre-exposure to heliox if exposure durations were less than 1 min, but were relatively constant with exposures greater than 1 min. (B) Residual helium in empty chamber fell with flush time duration. (C) Helium yield from the animal increased until wash-out times exceed 2 min.