Direct animal calorimetry, the underused gold standard for quantifying the fire of life

Abstract

Direct animal calorimetry, the gold standard method for quantifying animal heat production (HP), has been largely supplanted by respirometric indirect calorimetry owing to the relative ease and ready commercial availability of the latter technique. Direct calorimetry, however, can accurately quantify HP and thus metabolic rate (MR) in both metabolically normal and abnormal states, whereas respirometric indirect calorimetry relies on important assumptions that apparently have never been tested in animals with genetic or pharmacologically-induced alterations that dysregulate metabolic fuel partitioning and storage so as to promote obesity and/or diabetes. Contemporary obesity and diabetes research relies heavily on metabolically abnormal animals. Recent data implicating individual and group variation in the gut microbiome in obesity and diabetes raise important questions about transforming aerobic gas exchange into HP because 99% of gut bacteria are anaerobic and they outnumber eukaryotic cells in the body by ~10-fold. Recent credible work in non-standard laboratory animals documents substantial errors in respirometry-based estimates of HP. Accordingly, it seems obvious that new research employing simultaneous direct and indirect calorimetry (total calorimetry) will be essential to validate respirometric MR phenotyping in existing and future pharmacological and genetic models of obesity and diabetes. We also detail the use of total calorimetry with simultaneous core temperature assessment as a model for studying homeostatic control in a variety of experimental situations, including acute and chronic drug administration. Finally, we offer some tips on performing direct calorimetry, both singly and in combination with indirect calorimetry and core temperature assessment.

Direct calorimetry identifies deficiencies in respirometry for the determination of resting metabolic rate in C57Bl/6 and FVB mice

“…The most important among these are that (5) 1) test animals utilize fuels with the same stoichiometry as farm animals studied in the original experiments from 1900 to 1940, 2) net substrate interconversion (e.g., lipogenesis from glucose, gluconeogenesis from protein, or ketogenesis from triglycerides) is negligible and constant, 3) the total body CO 2 pool is constant (e.g., normal and stable blood pH and bicarbonate), 4) energy transfer from protein oxidation is low, constant, and specifiable, and 5) anaerobic metabolism is negligible. One may reasonably expect that genetically modified mice, which are nearly ubiquitous in modern experimental physiology and pharmacology (and especially those models with direct modifications to mitochondrial function), will violate at least one of these many assumptions (5).…”

“…Based on empirical data from chemical reactions performed in the 1920s (7), an equation relating gas exchange to heat production was constructed, and this equation is used by most major commercially available calorimetry systems. The validity of respirometry results depends on a series of untested and/or untestable assumptions (5,6,8). The most important among these are that (5) 1) test animals utilize fuels with the same stoichiometry as farm animals studied in the original experiments from 1900 to 1940, 2) net substrate interconversion (e.g., lipogenesis from glucose, gluconeogenesis from protein, or ketogenesis from triglycerides) is negligible and constant, 3) the total body CO 2 pool is constant (e.g., normal and stable blood pH and bicarbonate), 4) energy transfer from protein oxidation is low, constant, and specifiable, and 5) anaerobic metabolism is negligible.…”

Calorespirometry reveals that goldfish prioritize aerobic metabolism over metabolic rate depression in all but near-anoxic environments

“…Addink et al, 1991;van Waversveld et al, 1989;van Ginneken et al, 2004), something not all fish species are capable of (Stangl and Wegener, 1996). We used calorespirometry because it is the 'gold standard' of MR measurements and the only way to accurately measure MR on hypoxemic animals in real time (see Kaiyala and Ramsay, 2011;Nelson, 2016). Despite the superiority of calorespirometry, only a few studies have measured the metabolic heat of fishes (Addink et al, 1991;Regan et al, 2013;Stangl and Wegener, 1996;van Ginneken et al, 1994van Ginneken et al, , 1997van Ginneken et al, , 2004van Waversveld et al, 1989), and only three of these (van Ginneken et al, 1994(van Ginneken et al, , 1997(van Ginneken et al, , 2004 have measured metabolic heat at Pw O2 other than normoxia and anoxia.…”

A simple and affordable calorespirometer for assessing metabolic rates of fishes

“…This is most evident in cases of anoxia-tolerant organisms like the painted turtle (Chrysemya picta), crucian carp (Carassius carassius) and goldfish (Carassius auratus auratus), where attempts to quantify metabolic rate via respirometry in anoxia are futile because of the organism's complete reliance on anaerobic processes to support energy turnover. Like aerobic pathways, however, these pathways yield heat as a by-product, and the total amount of heat lost by an animal to its environment is proportional to its total energy turnover (minus that conserved in carbon bonds) (Mendelsohn, 1964;Mclean and Tobin, 1987;Kaiyala and Ramsay, 2011). Measuring this heat via calorimetry is therefore an effective way of estimating an animal's metabolic rate in situations where aerobic metabolism may be compromised.…”

“…Despite direct animal calorimetry being the 'gold standard for quantifying the fire of life' (Kaiyala and Ramsay, 2011), it is a seldom-used technique owing to its reputed difficulty and expense when compared with respirometry. These difficulties are especially true when working with ectothermic animals like fish, whose lower metabolic rates produce less heat compared with similarly sized endotherms.…”