INTRODUCTION

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THE PHENOMENON of hypometabolism during hypoxia is a common occurrence in many medium- and small-sized mammalian species, and it is particularly evident in the young and newborn (22). Several experiments have indicated that the phenomenon results largely from the inhibition of thermogenic processes (11, 22) and therefore is more pronounced in the cold. The mechanisms responsible for the hypoxic drop in O₂ consumption (O₂) are unclear. The simplest possibility is that of a limitation in the availability of O₂ to the mitochondria. At least in adult mammals, this seems extremely unlikely in view of 1) the fact that the hypometabolic response can occur with mild degrees of hypoxemia and 2) the enormous affinity for O₂ of mitochondrial respiratory enzymes (17). The observations that not only the mechanical events but also the electrical activity of shivering muscles are depressed by hypoxia (13) and that, during hypoxic hypometabolism, O₂ can be raised by exposure to cold (12) or by the administration of mitochondrial uncouplers (28) all indicate that the hypoxic decrease in O₂ is not the result of a limitation in O₂ supply to the tissues.

In the hypoxic newborn, in contrast to the adult, the possibility of O₂ being limited by the availability of O₂ has not been positively excluded. In favor of such a possibility is the fact that, in the newborn, the hyperpneic response to hypoxia is small or absent (20), suggesting that the pulmonary convection system is not as effective as in adults. In addition, in several newborn species, including human infants, breathing a hyperoxic gas raises O₂. This could be interpreted as indicating the presence of O₂ availability limitation even in normoxia (22). On the other hand, several observations in neonatal animals contradict such a possibility. For example, hypoxic hypometabolism often occurs with minimal or no sign of O₂ debt or lactic acidemia (5, 8). Furthermore, experimental interventions, such as an increase in body temperature (Tb) or preexposure to chronic hypoxia, resulted in an increase in ventilation and in arterial O₂ content (Ca_O2), respectively, but did not modify the hypoxic drop in O₂ (9, 25, 27). These observations would support the concept of a controlled and regulated inhibition of energy metabolism during hypoxia.

The present study was performed on conscious newborn dogs breathing gas mixtures of progressively lower O₂ concentrations (FI_O2) under warm or cold conditions. We reasoned that during hypoxic hypometabolism similar FI_O2-O₂ relationships between these two conditions would be consistent with the possibility of O₂ being limited by O₂ supply. This proved not to be the case. Further analysis of the parameters of blood oxygenation was compatible with the hypothesis that the drop in O₂ during hypoxia is a regulated response.

	METHODS

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Experiments were conducted on a total of 27 conscious newborn dogs (ages 1-2 wk) after approval of the experimental protocol by the Animal Care Committee of McGill University.

Early on the day of experimentation, 13 puppies (weight, 675 ± 20 g; age, 11 ± 1 days) were instrumented while they were anesthetized with halothane. The left common carotid artery and right internal jugular vein were exposed through a midline incision in the neck. Through these vessels, catheters were placed in the aorta and in the superior vena cava, and the incision was closed with sutures. Colonic temperature, taken as representative of Tb, was continuously monitored via a thermocouple probe placed through the rectum. The puppy was allowed to recover for 4 h. During this period, Tb was maintained at 37.5°C by adjusting radiant heat to achieve an ambient temperature (Ta) of ~35°C immediately after the intervention, then decreasing to ~25°C when the animal was fully awake. The puppy also received a continuous intravenous infusion of 10% dextrose in water at a rate of 150 ml · kg¹ · day¹. All animals were fully awake within 4 h of the termination of anesthesia; experiments were then begun.

The animal was placed unrestrained in a body plethysmograph that consisted of a cylindrical chamber measuring 9 × 30 cm, through which constant air flow, at the rate of 1.8 l/min, was maintained by a calibrated flowmeter. The chamber was inside a larger container that acted as a water bath to control the chamber's Ta at 30 or 20°C. The chamber's Ta was continuously monitored by tungsten-constantan thermocouples. To allow for the collection of blood samples, the arterial and venous catheters were exteriorized through small openings in the chamber. The catheters were connected to Hewlett-Packard pressure transducers for continuous measurement of systemic arterial pressure and central venous pressure.

Arterial blood gases, arterial O₂ saturation (Sa_O2), superior vena cava O₂ saturation [taken as index of mixed venous O₂ saturation (_O2); Ref. 10], as well as hemoglobin concentration (Hb, g/100 ml) were determined from 200 µl arterial and venous blood samples by using an Instrumentation Laboratories model 1302 blood-gas analyzer and a Radiometer model OSM2b hemoximeter; both were calibrated daily. For any _O2, mixed venous O₂ partial pressure (_O2) was extrapolated from the Sa_O2-arterial PO₂ (Pa_O2) relationship of the arterial samples. Blood-gas partial pressures were corrected to the Tb of the animal. A total of <3 ml of blood was taken from each animal. Ca_O2 and mixed venous blood O₂ content (_O2) were calculated (in ml O₂ /100 ml blood) from the corresponding O₂ saturation and Hb [(saturation/100) · Hb · 1.34 = ml O₂ /100 ml blood].

O₂ and CO₂ production (CO₂) were measured by a flow-through method (7), with the use of calibrated Beckman OM-11 and LB-2 gas analyzers, and the values were converted to STPD conditions. Cardiac output was calculated by using the Fick principle (O₂/arterial  venous O₂ content), and alveolar ventilation (A) was computed from the alveolar gas equation for CO₂ [A = (CO₂/PA_CO2) · Pb], with Pb representing dry barometric pressure and alveolar CO₂ partial pressure (PA_CO2) considered equal to the arterial value.

Once the animal was resting quietly in the metabolic chamber at the desired Ta, the experimental protocol consisted of sequential exposures to FI_O2 of 21, 18, 15, 12, 10, 8, and 6%. Measurements were obtained at the end of each exposure, which lasted 15 min. Seven animals were studied under warm conditions (Ta = 30°C), and six were studied in cold conditions (Ta = 20°C). There were no significant differences between these two groups with respect to age (both groups, 11 ± 1 days old) or weight (warm group, 690 ± 36 g; cold group, 657 ± 13 g). At the end of the experiment, the animals were killed with an intravenous injection of pentobarbital sodium, and the correct placement of the catheters was verified.

In a separate group of 14 puppies, O₂ was measured after the protocols described above, without prior anesthesia or instrumentation. These animals were taken from their mother shortly before placement in the metabolic chamber and returned to her immediately after the experiment. Hence, it was possible to study these animals under both warm and cold conditions, on separate days, and in random order. There was no significant difference in their ages or weights when studied under warm conditions (7 ± 0.2 days, 450 ± 30 g) or cold conditions (7 ± 0.7 days, 486 ± 34 g).

All data are expressed as means ± SE. Significant differences between two groups of data were evaluated by a two-tailed t-test. For both warm and cold conditions the FI_O2-O₂ relationship was analyzed by analysis of variance with repeated measurements, followed by six post hoc Bonferroni's limitations, to establish at which FI_O2 the O₂ differed significantly from that in air. Regression lines were calculated for all the O₂ data points that were significantly below the normoxic O₂. Significant differences in the slope of these relationships between the warm and cold groups were assessed by a two-tailed t-test. In all cases, the null hypothesis of no effect was rejected at P < 0.05.

	RESULTS

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Instrumented vs. noninstrumented puppies. The FI_O2-O₂ relationships of the instrumented animals studied under warm (n = 7) and cold (n = 6) conditions, as well as those of the noninstrumented animals studied in both conditions (n = 14), are presented in Fig. 1. In the cold, the O₂ in normoxia was ~70% greater than in warm conditions, averaging 25.5 ± 1.4 vs. 15.0 ± 0.8 ml · kg¹ · min¹, respectively. During mild hypoxia, O₂ was only minimally affected, but it decreased progressively as the hypoxia was made more severe. There was no significant difference between the FI_O2-O₂ relationship of the instrumented and noninstrumented puppies.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   O2 consumption (O2) in conscious puppies at various inspired O2 concentrations (FIO2) in warm (30°C) and cold (20°C) conditions. Animals were either intact (n = 14 for each condition) or previously instrumented (n = 6 in cold, n = 7 in warm). Values are means ± SE. Instrumentation did not alter response to either cold or hypoxia.

Normoxia. In the cold, O₂ was increased and Tb was slightly decreased compared with those measurements in the warm animals. The higher O₂ in the cold group was entirely achieved by a greater O₂ extraction, with no change in cardiac output. This was reflected by a greater difference in arteriovenous O₂ saturation and content in the cold animals (Table 1). The increase in O₂ was perfectly matched by the increase in A, with no change in arterial PCO₂ (Pa_CO2).

Hypoxia. Under warm conditions, a significant drop in O₂, compared with normoxia, occurred at FI_O2 of 10, 8, and 6%. In the cold condition, the drop was significant at FI_O2 of 12, 10, 8, and 6%. Thus, at the FI_O2 of 10%, the puppies were hypometabolic during both warm and cold conditions, although the O₂ was significantly higher in the cold animals compared with the warm animals (14.1 ± 0.9 vs. 11.4 ± 0.5 ml · kg¹ · min¹, respectively; P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Arterial partial pressure of O2 (PaO2), arterial O2 content (CaO2), and arterial partial pressure of CO2 (PaCO2) at various FIO2 during warm (open symbols; n = 7) and cold conditions (solid symbols; n = 6). Symbols indicate mean values; bars, SE.

Oxygenation-O₂ relationships during hypometabolism. The regression lines between various parameters pertinent to blood oxygenation or O₂ transport and O₂ were calculated for all FI_O2 values with significant hypometabolism (i.e., at FI_O2 of 12, 10, 8, and 6% in the cold and at FI_O2 of 10, 8, and 6% in warm conditions). The slopes of these functions were significantly less in warm than in cold conditions, with the exception of the Pa_O2-O₂ relationships, for which the difference in slope did not reach statistical significance (Table 2). The relationships between Ca_O2 or P and O₂ are represented by the continuous lines in Figs. 3 and 4.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   CaO2 and O2 in warm and cold conditions. Symbols are group means while breathing at progressively lower FIO2 (values in parentheses). Bars, SE. Oblique lines are linear regressions through CaO2-O2 data points, with O2 values significantly below normoxia; i.e., at FIO2 of 10, 8, and 6% in warm condition, 12, 10, 8, and 6% in cold condition.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Mixed venous partial pressure of O2 vs. O2 in warm and cold conditions. Symbols are group means while breathing at progressively lower FIO2 (values in parentheses). Bars, SE. Oblique lines are linear regressions through data points with O2 values significantly below normoxia; i.e. at FIO2 of 10, 8, and 6% in warm conditions, and 12, 10, 8, and 6% in cold conditions.

	DISCUSSION

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In mammals, the hypometabolic response to hypoxia is accompanied by inhibition of the shivering and nonshivering components of thermogenesis. Thus, in the cold condition, hypometabolism during hypoxia is more pronounced and occurs at a higher FI_O2 value than in warm conditions. This also occurred in the present experiments, in agreement with numerous previous reports on adults and newborns of various species (see Refs. 11 and 22 for reviews). Among adult mammals, hypoxic hypometabolism is a common occurrence in species of medium and small sizes (7). The hypoxic O₂ can be increased by cold exposure (12) or by pharmacological interventions (28). This fact indicates that the hypometabolic response to hypoxia is a regulated phenomenon not limited by the availability of O₂.

In the newborn mammal, some observations can be considered to support the possibility that, during hypoxia, O₂ is limited by the availability of O₂. During normoxic breathing, newborns often show some degree of hypoxemia, and breathing hyperoxic gases raises their resting O₂ (22). These phenomena have been interpreted as an indication that neonatal O₂ is limited by O₂ availability (23). The site of this limitation might be anywhere along the O₂ cascade, including the pulmonary convection mechanisms, because the hypoxic hyperpnea of newborns is most often less marked than in adults (20). On the other hand, other observations are not compatible with the idea of hypoxic O₂ in neonates being limited by O₂ supply. For example, in newborn rats exposed to cold, the maximal mass-normalized O₂ attained during hyperoxia was much less than that attained by adult rats in cold conditions (4). This suggests some limitation in the rate of O₂ utilization independent of O₂ availability. In addition, neither an increase in O₂-carrying capacity by preexposure to chronic hypoxia (9) nor an increase in minute ventilation by artificially raising Tb to the normoxic value (25) had any effect on the hypoxic hypometabolic response. Finally, the reports that hypoxic hypometabolism can occur without lactic acidemia or without a payment back of the "O₂ debt" on return to normoxia (1, 5, 8) cannot be easily reconciled with the possibility of an O₂-supply limitation being responsible for the hypoxic hypometabolism of newborns.

The results of the present experiments do not support the possibility of O₂ limitation being responsible for the hypoxic hypometabolism of newborns. In fact, hypometabolism under warm conditions occurred in association with levels of arterial and venous oxygenation that could sustain higher O₂ levels during cold exposure. This is demonstrated by comparison of the data obtained under warm and cold conditions at a FI_O2 of 10%, and by comparison of the regression lines for the relationships between various parameters pertinent to blood oxygenation or O₂ transport and O₂ during hypoxic hypometabolism. From data obtained by Hill in 1- to 26-day-old kittens exposed to progressively lower FI_O2 at 35 and at 22°C (see Fig. 7 of Ref. 15), it would seem clear that in warm conditions O₂ decreased when FI_O2 was 12%. However, the O₂ value was substantially lower than that attained at the same FI_O2 when the kittens were cold, in complete agreement with the present results in puppies (Fig. 1).

Living creatures can be schematically divided into "conformers" and "regulators" depending on their success in maintaining body homeostasis against external challenges (14, 26). The homeothermic characteristics of mammals and birds reflect their thermoregulatory abilities against changes in thermal conditions. This contrasts with the poikilothermic behavior of lower vertebrates and invertebrates. Similarly, the constancy of O₂ during decreases in FI_O2 would be an indication of O₂ regulation, whereas a drop in O₂ during environmental hypoxia would be a manifestation of O₂ conformism (19). It might be expected that the latter would be typical of organisms that are phylogenetically and ontogenetically less evolved. In reality, such a distinction has numerous exceptions. During hypoxia, many adult mammals decrease O₂, therefore conforming to O₂ availability (7), and even very simple organisms show some forms of regulation to hypoxia and not merely a reduction in energy production (14). The results of the present study further blur this distinction, indicating that the apparent O₂ conformism of the newborn is in reality a phenomenon of "regulated conformism," i.e., a metabolic adaptation not obligatorily dictated by the availability of O₂.

The mechanisms whereby O₂ is controlled during hypoxia are unknown. Carotid body afferent information is not essential for the hypometabolic response to hypoxia (2, 11). The occurrence of hypoxic hypometabolism during warm conditions, when the thermogenic demands are presumably minimal, is uncommon in the adult but has been observed previously in newborns (2, 15, 21, 31). This indicates that, in addition to thermogenesis, hypoxia inhibits other O₂-consuming processes in the neonate. In the newborn mammal, these likely include the energy-dependent processes required for the maintenance of skeletal muscle tone and activity, cell excitability, and tissue growth and differentiation (22). In addition, the cytosol has numerous O₂-dependent enzymes with a high Michaelis constant for O₂, i.e., low O₂ affinity (17), as opposed to the extremely low Michaelis constant of the mitochondrial respiratory enzymes. The cytosolic functions served by these enzymes therefore have the potential for sensing O₂ availability and thus controlling the hypometabolic response. The possibility of O₂ influencing cellular O₂, not as a substrate but rather as a regulatory molecule, has been recently discussed and has numerous plausible arguments (16). The hypothalamic nuclei, because of their diverse regulatory functions, including the control of nonshivering thermogenesis (24, 29, 30, 33), could be an appropriate site for the systemic integration of the hypometabolic response. In this regard, some hypothalamic neurons are specifically sensitive to hypoxia, independent of peripheral chemoinputs (3).

The hyperventilatory response to hypoxia was remarkably similar between warm and cold conditions, as shown by the essentially identical decrease in Pa_CO2. This indicates that the hypometabolic response to hypoxia, despite its large difference between warm and cold conditions, was perfectly matched by changes in A. This is consistent with the idea that metabolic rate plays an important role in determining the magnitude of the hyperpneic response to hypoxia (20). In cold conditions, Pa_O2 was slightly lower, at any given FI_O2, than in warm conditions (Fig. 2), presumably because of a small worsening of the ventilation-perfusion mismatch. However, Ca_O2 did not differ, because the 4-5°C drop in Tb shifted the O₂ dissociation curve to the left (Pa_O2-Ca_O2). Although the carotid bodies are known to respond to Pa_O2 rather than to Ca_O2 (6), the level of hyperventilation was the same under warm and cold conditions. Probably, the lower Tb in the cold decreased the ventilatory chemosensitivity (11, 18), hence offsetting the slightly higher hypoxic stimulus.

In conclusion, in conscious puppies, hypoxic hypometabolism during warm conditions was manifest at levels of arterial oxygenation that, when coupled with a cold stimulus, could sustain a higher O₂. Furthermore, during hypoxic hypometabolism under warm conditions, _O2, which can be equated to the diffusional pressure driving O₂ to the mitochondria (32), was not at its minimum, and, for the same level of oxygenation, cold stimulation raised O₂. Thus these results support the view that, during neonatal hypoxia, the drop in O₂ is a regulated process and not the result of limitation in the supply of O₂.