Measurement of 2H2O by IR absorbance in doubly labeled H2O studies of energy expenditure

The energy expenditure of animals in their natural surroundings can be determined by measuring the turnover in body water of isotopes of oxygen and hydrogen. We evaluated the use of infrared spectrophotometry for measuring 2H2O in small (20-microliters) water samples also labeled with 18O. For 2H2O over the enrichment range of 0.1-1 atom%, there was a linear relationship between infrared absorbance and 2H2O enrichment. 2H2O enrichments could be measured with a precision and accuracy of less than or equal to 1%, using this relationship. The presence of 18O in water samples in enrichments of up to 1 atom% had no significant effect on measurement of 2H2O by infrared absorbance. We measured the simultaneous turnover rates of 2H2O and 3H in mice and turtles also labeled with 18O. Our results validated the use of infrared absorbance in doubly labeled water measures of energy expenditure and indicated that the fractionation factors in vivo for 2H2O and 3H do not differ.