Measurements of 2 H and 18 O in body water: analytical considerations and physiological implications

Abstract

Measurement of energy expenditure with doubly-labelled water and of body composition and breast milk output with 2 H or 18 O requires accurate and precise techniques for measuring isotopic enrichments. The possibility of an inaccuracy in measurements of 2 H and 18 O isotopic enrichment arising from the matrix in biological fluids was investigated (1) by simulating a dilution experiment in both water and urine samples and (2) by reconstituting urine samples, ranging from 10 to 60 g/kg in solid concentration, from freeze-dried urinary solids mixed with either natural abundance or doubly-labelled water. Current techniques involved in measuring 2 H and 18 O isotopic enrichments were used (reduction of the samples to H 2 gas with either Zn or U, and CO 2 /H 2 O equilibration or direct measurement of mass 20:18 ratios on water vapour for 18 O analysis). All four methods accurately measured serial dilutions in both urine and water. Dilution space calculated from isotopic enrichments, compared with the water content of urine (determined by freeze-drying and accounting for exchangeable isotopes) was overestimated by about 2.4 % by the Zn technique whereas other methods were accurate. The urinary solids content of a water solution was related to that inaccuracy. The use of the Zn technique with biological samples is likely to create biases in 2 H distribution space. Examination of recent literature supports this view. Caution should therefore be used when physiological conclusions have to be made from the relative size of 2 H and 18 O distribution spaces.