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

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.     

Synchronous direct gradient layer and indirect room calorimetry

Rumpler, James L., and William V. Seale. Synchronousdirect gradient layer and indirect room calorimetry.J. Appl. Physiol. 83(5):1775-1781, 1997.A dual direct/indirect room-sized calorimeter isused at the Beltsville Human Nutrition Research Center to measure heatemission and energy expenditure in humans. Because the response timesof a gradient layer direct calorimeter and an indirect calorimeter arenot equivalent, the respective rate of heat emission and energyexpenditure cannot be directly compared. A system of equations has beendeveloped and tested that can correct the respective outputs of thedirect gradient layer calorimeter and indirect calorimeter for delaysdue to the response times of the measurement systems. Performance testsusing alcohol combustion to simulate a human subject indicate accurate measurements of heat production from indirect (99.9 ± 0.4%),indirect corrected for response time (99.9 ± 0.5%), direct (99.9 ± 0.8%), and direct corrected for response time (99.9 ± 0.8%)calorimetry systems. Results from 24-h measurements in 10 subjectsindicate that corrected heat emission is equivalent to (99.8 ± 2.0%) corrected energy expenditure. However, heat emission measuredduring sleep was significantly greater (14%) than energy expenditure,suggesting a change in the energy stored as heat in the body. Thisdifference was reversed during the day. These results illustrate howthe simultaneous measurement of heat emission and energy expenditure provides insights into heat regulation.

     

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N₂ bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0–8 h) and long-term severe hypoxia (8–72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.Abbreviations APW artificial pond water - HPLC high-performance liquid chromatography - fw fresh weight - HP heat production - HD heat dissipation - MR metabolic rate     

Dynamics of the heat exchange in rats upon getting out of the state of artificial hypothermia

With the help of thermometry and general calorimetry, changes in the heat exchange were determined in the non-hibernating mammals (rats) upon their getting out of artificial deep hypothermia (the temperature in the rectum was 22-24 degrees C) at the temperature in the calorimeter chamber 22 degrees C. An attempt was made to find out what part of the heal production during animal self-warming is used for increasing its body temperature and what part of the heat production is released to the environment by the animal. The experiments revealed a complex relationship between the body temperature, the heat loss, and the total heat production during the animal self-warming. The result was that the total heat production first increased and, after reaching the maximum, decreased gradually. The experiments showed that during two and more hours of observation the body temperature did not reach the starting level, the same was true for the total heat production, which was the sum of the heat loss and the heat production spent for warming the animal body.     

Dynamics of the heat exchange in rats upon getting out of the state of artificial deep hypothermia

With the help of thermometry and general calorimetry the changes in the heat exchange were determined in the rats upon their getting out of artificial deep hypothermia (the temperature in the rectum was 20 degrees C) at the temperature in the calorimeter chamber 20 degrees C. An attempt was made to find out what part of the heat production during an animal self-warming is used for increasing its body temperature and what part of the heat production is released to the environment by the animal. The experiments revealed a complex relationship between the body temperature, the heat loss, and the total heat production during the animal selfwarming. The total heat production first increased and, after reaching the maximum, decreased gradually. Moreover, the experiments showed that during three and more hours of observation the body temperature did not reach the initial level, the same was true for the total heat production, which in these experiments was the sum of the heat loss and the production spent for warming the animal body.     

Percent relative cumulative frequency analysis in indirect calorimetry: application to studies of transgenic mice

Indirect calorimetry is commonly used in research and clinical settings to assess characteristics of energy expenditure. Respiration chambers in indirect calorimetry allow measurements over long periods of time (e.g., hours to days) and thus the collection of large sets of data. Current methods of data analysis usually involve the extraction of only a selected small proportion of data, most commonly the data that reflects resting metabolic rate. Here, we describe a simple quantitative approach for the analysis of large data sets that is capable of detecting small differences in energy metabolism. We refer to it as the percent relative cumulative frequency (PRCF) approach and have applied it to the study of uncoupling protein-1 (UCP1) deficient and control mice. The approach involves sorting data in ascending order, calculating their cumulative frequency, and expressing the frequencies in the form of percentile curves. Results demonstrate the sensitivity of the PRCF approach for analyses of oxygen consumption (.VO2) as well as respiratory exchange ratio data. Statistical comparisons of PRCF curves are based on the 50th percentile values and curve slopes (H values). The application of the PRCF approach revealed that energy expenditure in UCP1-deficient mice housed and studied at room temperature (24 degrees C) is on average 10% lower (p < 0.0001) than in littermate controls. The gradual acclimation of mice to 12 degrees C caused a near-doubling of .VO2 in both UCP1-deficient and control mice. At this lower environmental temperature, there were no differences in .VO2 between groups. The latter is likely due to augmented shivering thermogenesis in UCP1-deficient mice compared with controls. With the increased availability of murine models of metabolic disease, indirect calorimetry is increasingly used, and the PRCF approach provides a novel and powerful means for data analysis.     

The anaerobic energy metabolism of goldfish determined by simultaneous direct and indirect calorimetry during anoxia and hypoxia

Summary The energy flow of the anaerobic metabolism of glodfish at 20°C during hypoxia and anoxia was studied by simultaneous direct and indirect calorimetry. During anoxia the heat production as determined by direct calorimetry (180 J · h^–1 · kg^–0.85) is reduced to 30% of the normoxic level (570 J · h^–1 · kg^–0.85), which is the same reduction as found previously. The patterns of substrate utilization are compared with previous results, where the anoxic pattern was established by simultaneous calorimetry without carbon dioxide measurements. The present results, which do include carbon dioxide measurements, show the same pattern: carbohydrate and protein as substrates and carbon dioxide, ethanol and fat as end products. The pattern of substrate utilization at low oxygen levels is a combination of the anoxic pattern with an aerobic component. During anoxia only 5% of the metabolizable energy is used for energy metabolism. Of the remaining part (metabolizable energy for production) 60% is converted into ethanol and 40% into fat. At two hypoxia levels the distribution of the metabolizable energy for production into ethanol and fat is the same.     

Calorimetric Studies of Behavior, Metabolism and Energetics of Sessile Intertidal Animals

SYNOPSIS: Behaviors to conserve water during intertidal exposureat the same time impair respiratory gas exchange, so that observedresponses to emersion may reflect compromises between theseincompatible needs. Behavioral isolation of the tissues fromair results in the complete or partial reliance on anoxic energymetabolism, which is most reliably measured directly as heatdissipation. Combined direct calorimetry and indirect calorimetry(respirometry) enable the partitioning of total metabolic heatdissipation into its aerobic and anoxic components, which mayvary according to physical and biological factors. The musselMytilus edulis is tolerant of anoxia and saves water and energyduring aerial exposure in its rocky intertidal habitat by closingits shell valves and becoming largely anoxic. Like most suspensionfeeders in this habitat, its compensation for reduced feedingtime involves energy conservation; there is little evidencefor energy supplementation such as increases in feeding rateor absorption efficiency. Ammonia production continues duringaerial exposure and is involved in acid-base balance in thehemolymph and mantle cavity fluid. Infaunal cockles (Cardiumedule) and mussels (Geukensia demissa) gape their shell valves,remain largely aerobic and have high rates of heat dissipationduring intertidal exposure, a response which appears relatedto the lower desiccation potential and exploitation of richertrophic resources in their soft-sediment habitats. The variableexpansion of the symbiotic sea anemone Anthopleura elegantissimareflects interaction among the responses to desiccation, irradianceand continued photosynthesis by its zooxanthellae during exposureto air.     

Academic genealogy and direct calorimetry: a personal account

Each of us as a scientist has an academic legacy that consists of our mentors and their mentors continuing back for many generations. Here, I describe two genealogies of my own: one through my PhD advisor, H. T. (Ted) Hammel, and the other through my postdoctoral mentor, Knut Schmidt-Nielsen. Each of these pathways includes distingished scientists who were all major figures in their day. The striking aspect, however, is that of the 14 individuals discussed, including myself, 10 individuals used the technique of direct calorimetry to study metabolic heat production in humans or other animals. Indeed, the patriarchs of my PhD genealogy, Antoine Lavoisier and Pierre Simon Laplace, were the inventors of this technique and the first to use it in animal studies. Brief summaries of the major accomplishments of each my scientific ancestors are given followed by a discussion of the variety of calorimeters and the scientific studies in which they were used. Finally, readers are encouraged to explore their own academic legacies as a way of honoring those who prepared the way for us.     

Thermal equilibrium responses in Guzerat cattle raised under tropical conditions

The literature is very sparse regarding research on the thermal equilibrium in Guzerat cattle (Bos indicus) under field conditions. Some factors can modify the physiological response of Guzerat cattle, such as the reactivity of these animals to handling. Thus, the development of a methodology to condition and select Guzerat cattle to acclimate them to the routine collection of data without altering their physiological response was the objective of the preliminary experiment. Furthermore, the animals selected were used in the main experiment to determine their thermal equilibrium according to the thermal environment. For this proposal, the metabolic heat production and heat exchange between the animal and the environment were measured simultaneously in the field with an indirect calorimetry system coupled to a facial mask. The results of the preliminary experiment showed that the respiratory rate could demonstrate that conditioning efficiently reduced the reactivity of the animals to experimental handling. Furthermore, the respiratory rate can be used to select animals with less reactivity. The results of the main experiment demonstrate that the skin, hair-coat surface and expired air temperature depend on the air temperature, whereas the rectal temperature depends on the time of day; consequently, the sensible heat flow was substantially reduced from 70 to 20 W m⁻² when the air temperature increased from 24 to 34 °C. However, the respiratory latent heat flow increased from 10 to 15 W m⁻² with the same temperature increase. Furthermore, the metabolic heat production remained stable, independent of the variation of the air temperature; however, it was higher in males than in females (by approximately 25%). This fact can be explained by the variation of the ventilation rate, which had a mean value of 1.6 and 2.2 L s⁻¹ for females and males, respectively.     

Respiration and heat production in the embryonic development of chickens

A marked increase in the rate of oxagen consumption and heat production, measured by the direct method, is observed in the chick embryogenesis. The intensity of respiration and heat production of the embryos decreases as the development proceeds. During development the data obtained by direct and indirect calorimetry diverge. This divergence, referred to as psi u-function, gradually decreases by the moment of hatching. Differences in the value of heat production and psi u-function were found in crosses of meat and egg directions, related to differences in the growth rate and productivity of adult fowl.     

Circadian change of heat loss in response to change in core temperature in rats

1. 1.|Circadian changes in heat production (M), heat loss (H), core temperature (T_c) and feeding activity (FA) of ad-lib fed rats were observed by direct and indirect calorimetry.

2. 2.|M, H and T_c showed a clear nocturnal increase associated with several discrete increases.

3. 3.|Whereas the slope of M vs T_c relation did not change appreciably within a day, the slope of H vs T_c or thermal conductance vs T_c relation tended to decrease at night, implying that the correlation between heat loss and body temperature is also a function of time of day in rats.

Explicit formulation of titration models for isothermal titration calorimetry

Isothermal titration calorimetry (ITC) produces a differential heat signal with respect to the total titrant concentration. This feature gives ITC excellent sensitivity for studying the thermodynamics of complex biomolecular interactions in solution. Currently, numerical methods for data fitting are based primarily on indirect approaches rooted in the usual practice of formulating biochemical models in terms of integrated variables. Here, a direct approach is presented wherein ITC models are formulated and solved as numerical initial value problems for data fitting and simulation purposes. To do so, the ITC signal is cast explicitly as a first-order ordinary differential equation (ODE) with total titrant concentration as independent variable and the concentration of a bound or free ligand species as dependent variable. This approach was applied to four ligand-receptor binding and homotropic dissociation models. Qualitative analysis of the explicit ODEs offers insights into the behavior of the models that would be inaccessible to indirect methods of analysis. Numerical ODEs are also highly compatible with regression analysis. Since solutions to numerical initial value problems are straightforward to implement on common computing platforms in the biochemical laboratory, this method is expected to facilitate the development of ITC models tailored to any experimental system of interest.     

Measurement of fetal heat production using differential calorimetry

These experiments were undertaken to measure heat production of fetal lambs in utero by using differential calorimetry. We used the principle that fetal heat production, H(fetus), can be calculated from measurements of base-line temperature difference between mother and fetus, delta T(fetus), heat introduced from an external source, H(heater), and the increase in body temperature, delta T(heater), that results, i.e., H(fetus) = H(heater) X delta T(fetus)/delta T(heater). We placed microheaters (1.8 mm diam) in the inferior vena cavae of eight near-term lambs and placed thermistors and catheters into maternal and fetal vessels and amniotic fluid. Five days later, fetal arterial temperature averaged 0.54 +/- 0.02 degrees C (SE) higher than maternal arterial temperature. When the heater was turned on to dissipate 29-103 cal/min, fetal temperature increased to approach 0.1-0.5 degrees C higher than control; the final temperature was estimated using the rate of increase during the first 20 min. Fetal heat production averaged 47.1 +/- 4.1 cal X min-1 X kg-1 during the warming phase in these lambs, which weighed 3.26 +/- 0.36 kg. This value would be 3-4% less if corrected for the increase in metabolic rate caused by heating, assuming a Q10 of 2.5. Fetal heating did not alter fetal heart rate, blood pressure, or blood gas values significantly, nor was hemolysis visible in plasma samples. When heat production was calculated from the decrease in fetal temperature after the heater was turned off, an average value of 41.2 +/- 2.5 cal X min-1 X kg-1 was found. Because this value is comparable to the heating phase, fetal metabolic rate and the insulating properties of the fetal shell are not likely to have been changed by the heating.     

Supplemental ascorbic acid does not affect inferred heat loss in broiler chickens exposed to elevated temperature

The provision of supplemental ascorbic acid has been reported to lower the body temperature of chickens maintained at elevated environmental temperatures. Since body temperature is the net effect of heat production and heat loss, it is not known if the reductions in body temperature were due to a lower heat production or an increase in heat loss. The purpose of this work was to determine if supplemental ascorbic acid facilitates heat loss in chickens exposed to an elevated temperature. On day 12 post-hatch broiler chickens were implanted intra-abdominally with a thermo-sensitive radio transmitter. The following day, birds were placed inside an indirect calorimeter maintained at 34 C for 24 h and provided water containing 0 or 400 ppm ascorbic acid. Oxygen consumption, carbon dioxide production, heat production, respiratory exchange ratio, and body core temperature were measured for 3 h; beginning 21 h after the birds were placed inside the calorimeter. No differences were observed in heat production or body core temperature between birds provided or not and 400 ppm ascorbic acid. This suggests that ascorbic acid has no effect on heat loss. Birds provided ascorbic acid did exhibit a significantly lower respiratory exchange ratio suggesting a greater utilization of lipid for energy production. Although lipid has a lower heat increment compared with protein and carbohydrate, the significance of this finding to birds exposed to elevated temperature is not known. In conclusion, under the conditions of this study the provision of supplemental ascorbic acid to broiler chickens maintained at an elevated temperature did not affect heat loss as inferred from measured heat production and body core temperature.     

Interactions between substrate,fermentation end-products,buffering systems and gas production upon fermentation of different carbohydrates by mixed rumen microorganisms in vitro

Summary In animal nutrition, incubation of feed samples with CO₂/HCO₃-buffered rumen fluid is used to predict the nutritional values of the feed. During fermentation, volatile fatty acids (VFAs) are produced, which release CO₂ from the buffer through their H⁺ ions. This indirect gas production amounted to 20.8 ml gas per mmol VFA. By incubating glucose, rice starch and cellulose, the relationship between direct and indirect gas production in relation to fermentation kinetics was studied. The total amount of gas formed was found to be dependent on the composition of the fermentation end-products formed. This could be described by: ml gas = M_v·mmol HAc + 2M_v·mmol HB + 0.87M_v·mmol Tot. VFA where HAc = acetic acid; HB = butyric acid; and M_v = molar gas volume. No clear relationship was found between the rate of fermentation and total gas production. From rice starch more total gas was produced than from glucose and cellulose, which were fermented faster and slower, respectively. Correspondence to: S. F. Spoelstra     

Exercise and postexercise energy expenditure in growing pigs

Young pigs (ca. 10 kg) were trained to run on a motor-driven treadmill for 1 h each day. After a 2-week training period the gas exchange of exercised and control animals was measured using an open circuit, indirect calorimeter. The exercised pigs ran for 2 h in the calorimeter, and then rested for 2 h. They received a day's allocation of feed and remained in the calorimeter for a total of 23 h. The total heat production of the exercised pigs was 523 kJ/kg, compared with 433 kJ/kg of the controls. Monitoring the heat production throughout the 23-h period showed that only 43% of the extra heat dissipated by the exercised pigs was lost during the 2 h of exercise, with a higher rate of heat production for the remaining 21 h accounting for the 57% of the extra energy dissipated as heat. The results suggest that exercise increases energy expenditure well beyond the time devoted to the activity itself.     

A graphic analysis of respiratory heat exchange

A new graphic representation of respiratory heat exchange is proposed using the concept of equivalent temperatures directly related to enthalpy values. On such a diagram it is possible to 1) compute the value of the heat exchange (delta H) knowing the inspired temperature (TI) and the partial pressure of water vapor (PIH2O) [or the relative humidity (rhI)] of inspired gas; 2) estimate the variation in delta H following a given variation in TI and PIH2O or, inversely, to choose the variation in TI and PIH2O necessary to obtain a given variation in delta H; 3) dissociate inspiratory and expiratory exchanges and to evaluate the efficiency of the respiratory heat exchange process in different environmental situations; and 4) easily compare the results of different studies published on respiratory heat exchanges in humans or other animal species.     

Analysis of the Escherichia coli glucosamine-6-phosphate synthase activity by isothermal titration calorimetry and differential scanning calorimetry

Glucosamine-6-phosphate synthase (GlmS) is responsible for the first and rate-limiting step in the hexosamine biosynthetic pathway. It catalyzes the conversion of D-fructose-6P (F6P) into D-glucosamine-6P (GlcN6P) using L-glutamine (Gln) as nitrogen donor (synthase activity) according to an ordered bi-bi process where F6P binds first. In the absence of F6P, the enzyme exhibits a weak hydrolyzing activity of Gln into Glu and ammonia (glutaminase activity), whereas the presence of F6P strongly stimulates it (hemi-synthase activity). Until now, these different activities were indirectly measured using either coupled enzyme or colorimetric methods. In this work, we have developed a direct assay monitoring the heat released by the reaction. Isothermal titration calorimetry and differential scanning calorimetry were used to determine kinetic and thermodynamic parameters of GlmS. The direct determination at 37 °C of kinetic parameters and affinity constants for both F6P and Gln demonstrated that part of the ammonia produced by Gln hydrolysis in the presence of both substrates is not used for the formation of the GlcN6P. The full characterization of this phenomenon allowed to identify experimental conditions where this leak of ammonia is negligible. Enthalpy measurements at 25 °C in buffers of various heats of protonation demonstrated that no proton exchange with the medium occurred during the enzyme-catalyzed glutaminase or synthase reaction suggesting for the first time that both products are released as a globally neutral pair composed by the Glu carboxylic side chain and the GlcN6P amine function. Finally we showed that the oligomerization state of GlmS is concentration-dependent.     

Day-night variation of thermoregulatory responses to intraperitoneal electric heating in rats

1. 1.Hypothalamic temperature (T_hy), nonevaporative heat loss (R + C + K), evaporative heat loss (E), thermal conductance (k), metabolic heat production (M) and heat storage (S) of rats were simultaneously measured by direct and indirect calorimetry during internal heat loading (2 W per rat) with an intraperitoneal electric heater.

2. 2.The tests were made twice a day; once during the day (1000–1200 h) and once at night (2200–2400 h) at an ambient temperature of 24°C.

3. 3.The resting values of T_hy, colonic temperature, (R + C + K), E, M and heart rate, and the T_hy threshold for tail skin vasodilation (T_th) during internal heat load were significantly higher at night than during the day.

4. 4.The slopes showing the relationshiop between (R + C + K), k or M and T_hy were significantly steeper during the day than at night after T_hy exceeded T_th.

5. 5.The slopes showing the relationship between E or S and T_hy were not different during the day and at night.

6. 6.These results indicate that the responses of nonevaporative heat loss and heat production to internal heat load vary with the time of day in rats.