MSACL 2019 EU Abstract

Validation of the Doubly-labelled Water Method to Determine Total Energy Expenditure in a Rat Model

Anita Eberl (1), Simon Schwingenschuh (1), Albrecht Leis (2), Christoph Magnes (1)
(1) HEALTH – Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft mbH, Neue Stiftingtalstrasse 2, 8010 Graz, Austria; (2) JR-AquaConSol GmbH, Steyrergasse 21, 8010 Graz, Austria

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Anita Eberl (Presenter) Joanneum Research

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: The global increase in obesity is still one of the major health challenges, which drives the growing interest in understanding factors that influence total energy expenditure. Doubly-labelled water is commonly used to measure total energy expenditure in animals and humans. After a bolus dose of doubly-labelled water, hydrogen leaves the body primarily via water turnover, whereas oxygen is depleted both via water turnover and via CO2-exhalation. The excess disappearance rate of 18O relative to deuterium as a measure for the CO2-production rate is used as indirect measure of total energy expenditure.

Objectives: We aimed to validate the doubly-labelled water method to determine the total energy expenditure in a rat model using a metabolic cage, and to establish a sample preparation method suitable for low-volume plasma samples.
Methods: Intraperitoneal injection was used to administer a mixture of H218O and D2O in 6 rats housed in metabolic cages (PhenoMaster). We collected rat plasma samples over a period of 5 days to measure the total energy expenditure. Sample preparation was adjusted for low-volume rat plasma samples by investigating ultrafiltration sample preparations with 10, 30 and 50 kDa cut-off membranes. In addition to standard Isotope-Ratio-MS measurement, we used laser spectrometry to determine 18O and deuterium-enrichment. Rate constants and pool sizes were calculated using the slopes and intercepts of the log-transformed spectrometric data to determine the CO2-production.

Results: Total energy expenditure assessed by the doubly-labelled water method was in accordance with CO2-production data from the metabolic cage. Data showed that cut-off sizes of the ultrafiltration membranes did not influence measurement accuracy. Mass spectrometry and laser spectrometry data were well correlated independent of the used sample preparation method with correlation coefficients of 0.999 for both isotopes and a slope of 1.01 for deuterium and 0.98 for 18O.

Conclusion: Total energy expenditure measurements in rats by doubly-labelled water were validated using a metabolic cage and we successfully established a sample preparation method for low-volume plasma samples. The doubly-labelled water method can thus be used for reliable energy expenditure measurements covering typical observation times of one or more weeks in humans. The method is also a promising tool to assess the effect of routine activities or exercise on total energy expenditure in a home environment in obese patients.