Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry

This study describes methods and equipment for measurement of metabolic heat rates of cells and tissues under conditions that provide simultaneous determinations of the flux rates of both O2 and CO2. Isothermal measurement of metabolic heats are conducted in a sealed ampule. A trapping solution is employed to absorb metabolic CO2. Absorption of CO2 produces heat at a rate proportional to the rate of CO2 production. Under these conditions, O2 consumption by the tissue results in a decrease in the partial pressure of O2 within the sealed ampule. The decrease in pressure can be monitored with a pressure sensor and related to O2 consumption rates. The combined measurements of heat rates, CO2, and O2 fluxes provide important information on bioenergetic efficiency of cell metabolism. These data can also suggest possible shifts in metabolic pathways or substrate sources as cells develop, or are exposed to effectors, inhibitors, and environmental factors.     

Diurnal variation of heat intake in ovariectomized, steroid-treated rats

Ovariectomized rats trained to work for radiant heat reward in a cold environment were implanted with subcutaneous Silastic capsules containing either estradiol, progesterone, both estradiol and progesterone, or no hormone. The hormone treatments produced an average plasma estradiol concentration of 41 pg/ml and progesterone concentration of 20–50 ng/ml. All groups obtained more heat behaviorally when tested during the light phase of the LD cycle than when tested in the dark. Body temperatures and metabolic rates were higher during the night than during the day. There were no differences between groups in behavioral heat intake or body temperature. All hormone-treated groups showed a greater reduction in core temperature than the control group when an exogenous source of heat was not available, but there was no substantial effect of the hormone treatments on metabolic rate except for a 6–7% increase in metabolism of the estrogen group. The increased cooling rate of all hormone-treated groups may indicate a nonspecific steroid-induced increase in heat loss in the cold. The diurnal variation in heat intake establishes the LD cycle as a significant variable in thermoregulatory behavior of the rat. Thus, behavioral heat intake is high during the day when metabolism and body temperature are low, and low at night when metabolism and body temperature are high in this nocturnal species.     

Minimal metabolic rate, what it is, its usefulness, and its relationship to the evolution of endothermy: a brief synopsis

Minimal metabolic rate represents the minimal cost of living and appears to have the same relative composition of adenosine triphosphate processes in all organisms. Minimal metabolic rate is influenced by temperature and defines the standard metabolic rate (SMR) of animals. Animals that achieve SMR only for a given temperature are strictly ectothermic. Endotherms, on the other hand, are characterized by leakier membranes and an associated increase in cellular metabolism for a given temperature. The increase in cellular metabolism is coupled with an increase in heat production (i.e., obligatory thermogenesis) that, together with SMR, defines the basal metabolic rate of an endotherm. Consideration of minimal metabolic rate must take into account ecological and physiological processes, environmental influences, evolutionary arguments, and body size.     

Metabolic response of Platynota stultana pupae to controlled atmospheres and its relation to insect mortality response

The metabolic responses of Platynota stultana pupae to reduced O(2), elevated CO(2), and their combinations were investigated using microcalorimetry, and mortality of pupae under elevated CO(2) atmospheres was correlated with metabolic responses. The metabolic heat rate decreased slightly with decreasing O(2) concentration until a critical O(2) concentration (P(c)) below which the heat rate decreased rapidly. The P(c) increased with temperature. The percentage decreases of metabolic heat rate were comparable to the percentage decreases of O(2) consumption rate (RO(2)) at 10, 8, 6, and 4% O(2), but were smaller at 2 and 1% O(2). The metabolic heat rate decreased rapidly at 20% CO(2) relative to 0% CO(2), with little to no further decrease between 20 and 79% CO(2). The percentage decreases of RO(2) under 20 and 79% CO(2) at 20 degrees C were comparable to the percentage decreases of metabolic heat rates. The additive effects of subatmospheric O(2) and elevated CO(2) levels on reducing metabolic heat rate were generally fully realized at combinations of /=4% O(2), but became increasingly overlapped as the O(2) concentration decreased and the CO(2) concentration increased. The high susceptibility of pupae to elevated CO(2) at high temperature was correlated with high metabolic heat rate. The metabolic responses of pupae to reduced O(2) concentrations included metabolic arrest and anaerobic metabolism. The net effect of elevated CO(2) on the pupal respiratory metabolism was similar to that of reduced O(2); however, mechanisms other than the decrease of metabolism were also contributing to the toxicity of CO(2).     

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     

Skeletal muscle metabolism during exercise is influenced by heat acclimation

The influence of heat acclimation on skeletal muscle metabolism during submaximal exercise was studied in 13 healthy men. The subjects performed 30 min of cycle exercise (70% of individual maximal O2 uptake) in a cool [21 degrees C, 30% relative humidity (rh)] and a hot (49 degrees C, 20% rh) environment before and again after they were heat acclimated. Aerobic metabolic rate was lower (0.1 l X min-1; P less than 0.01) during exercise in the heat compared with the cool both before and after heat acclimation. Muscle and plasma lactate accumulation with exercise was greater (P less than 0.01) in the hot relative to the cool environment both before and after acclimation. Acclimation lowered (P less than 0.01) aerobic metabolic rate as well as muscle and plasma lactate accumulation in both environments. The amount of muscle glycogen utilized during exercise in the hot environment did not differ from that in the cool either before or after acclimation. These findings indicate that accumulation of muscle lactate is increased and aerobic metabolic rate is decreased during exercise in the heat before and after heat acclimation; increased muscle glycogen utilization does not account for the increased muscle lactate accumulation during exercise under extreme heat stress; and heat acclimation lowers the aerobic metabolic rate and muscle and blood lactate accumulation during exercise in a cool as well as a hot environment.     

A calorimetric investigation of the growth of the luminescent bacteria Beneckea harveyi and Photobacterium leiognathi.

Direct calorimetric determinations of the rate of heat production along with simultaneous determinations of the rate of photon emission and the number of viable cells have provided insight into the growth of Beneckea harveyi and Photobacterium leiognathi. These experiments were performed with a Tronac isothermal microcalorimeter modified with a fiber optic light guide to allow in situ detection of light. Escherichia coli and a dark variant of P. leiognathi were also examined to provide points of reference. It is demonstrated that B. harveyi seems to pause in the rate of metabolic heat production at the same point in time that the enzyme luciferase begins to be synthesized. This effect is not removed if B. harveyi is grown in conditioned medium. The thermograms for all species are correlated with cell generation time. The heat production per cell indicates that uncrowded cultures produce more heat than older, more crowded cultures, supporting the original observation of Bayne-Jones and Rhees (1929). These observations reopen for examination the suggestion that living systems tend toward a state of minimum metabolism per unit mass.     

Human heat balance during postexercise recovery: separating metabolic and nonthermal effects

Previous studies report greater postexercise heat loss responses during active recovery relative to inactive recovery despite similar core temperatures between conditions. Differences have been ascribed to nonthermal factors influencing heat loss response control since elevations in metabolism during active recovery are assumed to be insufficient to change core temperature and modify heat loss responses. However, from a heat balance perspective, different rates of total heat loss with corresponding rates of metabolism are possible at any core temperature. Seven male volunteers cycled at 75% of Vo(2peak) in the Snellen whole body air calorimeter regulated at 25.0 degrees C, 30% relative humidity (RH), for 15 min followed by 30 min of active (AR) or inactive (IR) recovery. Relative to IR, a greater rate of metabolic heat production (M - W) during AR was paralleled by a greater rate of total heat loss (H(L)) and a greater local sweat rate, despite similar esophageal temperatures between conditions. At end-recovery, rate of body heat storage, that is, [(M - W) - H(L)] approached zero similarly in both conditions, with M - W and H(L) elevated during AR by 91 +/- 26 W and 93 +/- 25 W, respectively. Despite a higher M - W during AR, change in body heat content from calorimetry was similar between conditions due to a slower relative decrease in H(L) during AR, suggesting an influence of nonthermal factors. In conclusion, different levels of heat loss are possible at similar core temperatures during recovery modes of different metabolic rates. Evidence for nonthermal influences upon heat loss responses must therefore be sought after accounting for differences in heat production.     

Global metabolomic responses of Escherichia coli to heat stress

Microbial metabolomic analysis is essential for understanding responses of microorganisms to heat stress. To understand the comprehensive metabolic responses of Escherichia coli to continuous heat stress, we characterized the metabolomic variations induced by heat stress using NMR spectroscopy in combination with multivariate data analysis. We detected 15 amino acids, 10 nucleotides, 9 aliphatic organic acids, 7 amines, glucose and its derivative glucosylglyceric acid, and methanol in the E. coli extracts. Glucosylglyceric acid was reported for the first time in E. coli. We found that heat stress was an important factor influencing the metabolic state and growth process, mainly via suppressing energy associated metabolism, reducing nucleotide biosynthesis, altering amino acid metabolism and promoting osmotic regulation. Moreover, metabolic perturbation was aggravated during heat stress. However, a sign of recovery to control levels was observed after the removal of heat stress. These findings enhanced our understanding of the metabolic responses of E. coli to heat stress and demonstrated the effectiveness of the NMR-based metabolomics approach to study such a complex system.     

Energetically costly behaviour and the evolution of resting metabolic rate in insects

1. A general hypothesis is presented to explain interspecific differences in size-independent resting metabolic rate. This hypothesis is based on a presumed trade-off between a low resting metabolism and adaptations of metabolism during activity.
2. With such a trade-off, selection to reduce resting metabolism is less intense in active species than in species where resting metabolism constitutes a large proportion of the daily metabolic costs. Those animals that spend more energy on activity should therefore have a higher resting metabolic rate than animals that spend less energy on activity.
3. A literature review reveals that flying insects have higher resting metabolic rates than species that use energetically less demanding types of locomotion.
4. Insects producing acoustic advertisement signals can be shown to have higher mass-independent resting metabolic rates than closely related species without this energetically demanding behaviour.
5. Literature data on vertebrate resting metabolic rates are also consistent with the presented hypothesis: the more energy animals spend on activity, the higher the mass-independent resting metabolic rate.     

Microcalorimetric study of the toxic effect of selenium on the mitochondrial metabolism of cyprinus carpio liver

The metabolic thermograms and heat output of mitochondria isolated from carp liver have been determined by using an LKB bioactivity monitor. The thermogram can be divided into four parts: the lag phase, active recovery phase, stationary phase, and decline phase. The thermokinetic equation was established for the active recovery and decline phase of metabolism as follows: dP/dt =k _mP (1-SP). The rate constantsk ₁ andk ₂ of two phases of active recovery and decline phase have been also calculated. The metabolism activity of mitochondrial inhibited by a high concentration of trace element selenium has been studied. The metabolic heat released, time of each phase, and rate constants can be significantly influenced by excess of selenite added. These results suggested that a high concentration of selenium can damage the structure and function of mitochondria, and thus influence their metabolism.     

Normoxic and anoxic heat output of the freshwater bivalves Pisidium and Sphaerium

In fasting Pisidium amnicum and Sphaerium corneum, regular periods of behavioural and metabolic quiescence were shown to occur in the normoxic, constant environment of the flow-through chamber of a heat-flow microcalorimeter. The metabolic rate was suppressed to 7.5% of normal at 10° C and to 8.5–9.7% at 20° C for periods exceeding the period of active metabolism by a factor of 3.5 at 10° C and 8.3 at 20° C. The rate of heat output during normoxic quiescence was equal to that during environmental anoxia, suggesting spontaneous achievement of body anoxia by complete shell closure. The mass-specific integrated heat output during closure periods was independent of size. Parallel observations on clam behaviour suggested that metabolic quiescence coincided with shell closure, and bursts of heat flow with active ventilation. Shell closure was accompanied by pronounced bradycardia, down to 20% of the active rate. In a constant environment, the rhythmic quiescence is regulated by shell closure which is probably triggered by lack of food. Regular quiescence of fasting bivalves may conserve energy reserves considerably, the amount depending on the possible excretion rate of the end products, and the post-quiescence recovery costs, which were not measured. Heat output during the active period was close to the average metabolic rate found earlier for Sphaeriidae. However, all the values determined so far are likely to be underestimates of the natural metabolism because the effects of digestion and growth are not included.     

Physiological responses in a variable environment: relationships between metabolism, hsp and thermotolerance in an intertidal-subtidal species

Physiological responses to temperature reflect the evolutionary adaptations of organisms to their thermal environment and the capability of animals to tolerate thermal stress. Contrary to conventional metabolism theory, increasing environmental temperatures have been shown to reduce metabolic rate in rocky-eulittoral-fringe species inhabiting highly variable environments, possibly as a strategy for energy conservation. To study the physiological adaptations of an intertidal-subtidal species to the extreme and unpredictable heat stress of the intertidal zone, oxygen consumption rate and heat shock protein expression were quantified in the sea cucumber Apostichopus japonicus. Using simulate natural temperatures, the relationship between temperature, physiological performance (oxygen consumption and heat shock proteins) and thermotolerance were assessed. Depression of oxygen consumption rate and upregulation of heat shock protein genes (hsps) occurred in sequence when ambient temperature was increased from 24 to 30°C. Large-scale mortality of the sea cucumber occurred when temperatures rose beyond 30°C, suggesting that the upregulation of heat shock proteins and mortality are closely related to the depression of aerobic metabolism, a phenomenon that is in line with the concept of oxygen- and capacity-limited thermal tolerance (OCLTT). The physiologically-related thermotolerance of this sea cucumber should be an adaptation to its local environment.     

Heat-transfer relations of avian nestlings

1. 1.|To determine the thermoregulatory prowess of altricial nestlings, we conducted both equilibrium and transient analyses of white-crowned sparrow nestings, a representative fringillid.

2. 2.|For an individual nestling at thermal equilibrium, feather development is the major factor reducing heat loss after 2 days of age; tissue- and boundary-layer resistances are of minor importance.

3. 3.|The nest substantially reduces wind speeds near the nestlings. Heat transfer through the nest material is of only moderate importance. Evaporation also appears to be a small proportion of total heat loss during hypothermia in natural environments.

4. 4.|Net long-wave radiant exchange is also minor, but short-wave radiation is potentially a major component of the nestling's energy budget, approaching the magnitude of maximal metabolic heat production.

5. 5.|When nestlings cool, their body mass and metabolic rate are also major importance in determining the rate of cooling, and (for metabolism) the equilibrium temperature as well.

6. 6.|The huddling together of nestlings is perhaps the single most important factor affecting heat transfer.

7. 7.|An older brood actually has more insulation than does an adult in the same microclimate.

Energetics of East African Pycnonotids1

Rate of oxygen consumption was measured in five bulbuls (Family Pycnonotidae) from western Uganda to evaluate whether this group is indeed characterized by the very low basal rates of metabolism previously reported. For three of these species, body temperature and rate of metabolism were measured as a function of ambient temperature from 10°C to 35°C. In these species body temperature was highly variable, and declined with ambient temperature in Andropadus virens. Such variation, in conjunction with behavioral adjustments, may reduce heat loss at low ambient temperatures. Body mass accounted for 98 percent of the variation in the basal rates of metabolism presented here. Basal rates in these species ranged from 81 to 90 percent of values predicted by the Aschoff–Pohl relationship for passerines, whereas previous measurements ranged from 56 to 72 percent of predicted values. This difference may reflect differences in species or measurement techniques, which, if the latter, suggests that the reduction in metabolic rate in this family may be less than originally thought. These data underline the importance of continued data collection on the metabolism of tropical birds, few of which have been measured to date.     

Foliar metabolic heat rate of seedlings and mature trees of Pinus ponderosa exposed to acid rain and ozone

We investigated the effects of acid rain and ozone on respiration rates of 1-year-old and current-year foliage of half-sib seedlings and mature clones of a ponderosa pine genotype by measurement of foliar metabolic heat rates. Two rain regimes (pH 5-1 and 3-0) were applied weekly to foliage only, from January to April 1992. Two ozone regimes (ambient and twice-ambient) were applied from September 1991 to November 1992. Metabolic heat rate was measured in April on 1-year-old foliage, in June on both 1-year-old and current-year foliage, and in November on current-year foliage in 1992. Except for current-year foliage in June, the metabolic heat rate was calculated per unit of both foliar dry mass and N mass. In seedlings, both measures of metabolic heat rate increased in late June for 1-year-old foliage exposed to twice-ambient ozone, and in November for current-year foliage exposed to the combination of twice-ambient ozone and pH 3-0 rain. In mature trees, metabolic heat rate was not affected significantly by ozone, rain acidity, or their interaction. In June, when both 1-year-old and current-year tissues were examined, the metabolic heat rate of expanding, current-year foliage was higher than that of fully expanded, 1-year-old foliage regardless of plant age or treatment combination.     

Colder environments did not select for a faster metabolism during experimental evolution of Drosophila melanogaster

The effect of temperature on the evolution of metabolism has been the subject of debate for a century; however, no consistent patterns have emerged from comparisons of metabolic rate within and among species living at different temperatures. We used experimental evolution to determine how metabolism evolves in populations of Drosophila melanogaster exposed to one of three selective treatments: a constant 16°C, a constant 25°C, or temporal fluctuations between 16 and 25°C. We tested August Krogh's controversial hypothesis that colder environments select for a faster metabolism. Given that colder environments also experience greater seasonality, we also tested the hypothesis that temporal variation in temperature may be the factor that selects for a faster metabolism. We measured the metabolic rate of flies from each selective treatment at 16, 20.5, and 25°C. Although metabolism was faster at higher temperatures, flies from the selective treatments had similar metabolic rates at each measurement temperature. Based on variation among genotypes within populations, heritable variation in metabolism was likely sufficient for adaptation to occur. We conclude that colder or seasonal environments do not necessarily select for a faster metabolism. Rather, other factors besides temperature likely contribute to patterns of metabolic rate over thermal clines in nature.     

Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (13)CO(2) dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.