Intensive Care Med (2001) 27: 1518±1525 DOI 10.1007/s001340101044
EXPERIM ENTAL
Increased systemic oxygen consumption offsets improved oxygen delivery during dobutamine infusion in newborn lambs
D. J. Penny T. Sano J. J. Smolich
Received: 7 September 2000 Final revision received: 21 June 2001 Accepted: 22 June 2001 Published online: 7 August 2001 Springer-Verlag 2001 This study was supported by a Grant-inAid from the National Heart Foundation of Australia and a Research Initiatives Grant from the Faculty of Medicine, Monash University. Data presented in part at The World Congress of Pediatric Intensive Care, Montreal, 2000
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D. J. Penny ´ T. Sano ´ J. J. Smolich ( ) Centre for Heart and Chest Research, Monash University Department of Medicine, Monash Medical Centre, Monash University, 246 Clayton Road, Clayton, Victoria, Australia, 3168 E-mail:
[email protected] Phone: +61-3-95 94 22 42 Fax: +61-3-95 94 62 39 D. J. Penny ´ J. J. Smolich Institute of Reproduction and Development, Monash University, Victoria, Australia D. J. Penny ´ T. Sano Department of Cardiology, Royal Children's Hospital, Victoria, Australia
Abstract Objective: To determine: 1) if dobutamine elicited a thermogenic response during postnatal development; and 2) if this response impacted on the balance between systemic O2 delivery (DO2) and O2 consumption (VO2), and involved one or a combination of adrenoceptor subtypes. Design: Prospective non-randomized unblinded study. Setting: University research laboratory. Subjects: Thirty-five Border-Leicester cross lambs used in a main study performed at 1±2 days (n = 7), 7±10 days (n = 7), and 6±8 weeks (n = 8), and in a adrenoceptor blockade substudy performed at 1±2 days (n = 13). Interventions: Lambs were instrumented under anaesthesia and dobutamine was infused at incremental rates of 1±40 mg/kg per minute. In separate subgroups of 1±2 day-old lambs, dobutamine was infused after selective or combined a1, b1, and b2adrenoceptor blockade. Measurements: Cardiac output, aortic and pulmonary arterial blood gases, and body temperature were measured. DO2 and VO2 were calculated. Main Results: Dobutamine increased DO2 similarly at all three ages. Dob-
utamine also increased VO2 in the absence of muscle shivering, but the average rise in 1±2 day-old lambs was sevenfold to 12-fold greater (P < 0.001) than in 7±10 day-old and 6±8 week-old animals, was associated with an increase in systemic O2 extraction, and accounted for » 90 % of the rise in DO2. Body temperature rose by 1.3 0.5 C in 1±2 dayold animals (P < 0.001), but was unchanged in 7±10 day-old or 6±8 week-old lambs. In 1±2 day-old lambs, rises in DO2, VO2, and body temperature induced by dobutamine were not affected by selective a1, b1 or b2 adrenoceptor blockade, but were markedly attenuated by combined adrenoceptor blockade. Conclusions: A substantial rise in VO2 which accompanied a pronounced thermogenic effect of dobutamine in newborn lambs utilized most of the associated increase in DO2 and appeared to be dependent on activation of multiple adrenoceptor subtypes. Keywords Adrenergic agonists ´ Adrenergic antagonists ´ Brown fat ´ Body temperature regulation ´ Oxygen consumption ´ Oxygen delivery
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Introduction Dobutamine, a synthetic catecholamine with predominant b1-adrenoceptor and additional a1- and b2-adrenoceptor-stimulating properties [1], is commonly used clinically as an inotrope [2, 3]. A major goal of inotropic therapy is to promote an environment conducive to adequate tissue oxygenation by increasing systemic O2 delivery (DO2) relative to changes in systemic O2 consumption (VO2). Thus, in adult humans [4] and experimental animals [5], dobutamine increases cardiac output and augments DO2. Because of a rise in tissue metabolism [2, 6], dobutamine also raises VO2, but this constitutes only » 10 % of the increment in DO2 [2, 4, 5]. Unlike the adult, however, little or no information is available about the effects of dobutamine on DO2, VO2, and associated oxygenation parameters in the newborn. This is of particular relevance because monitoring of peripheral oxygenation has been recommended as a useful adjunct in the clinical management of the critically ill neonate [7, 8, 9] and because the newborn has considerable capacity to generate heat via non-shivering thermogenesis (NST), a process which occurs in brown adipose tissue (BAT) [10], due to the presence of a unique mitochondrial `uncoupling protein' that uncouples oxidative phosphorylation, thereby producing heat rather than ATP during lipolysis [11]. NST can be activated by release of noradrenaline from sympathetic nerves within BAT [11] or infusion of this catecholamine [10] and is accompanied by substantial increases in systemic O2 extraction, VO2, and body temperature, as well as a fall in venous O2 content [10, 11, 12]. However, although dobutamine can activate lipolysis in isolated brown adipocytes [13, 14, 15], it is unknown if this inotrope promotes NST in the newborn and to what extent any accompanying rises in VO2 offset associated increases in DO2 and potential improvements in tissue oxygenation. To address these questions, this study evaluated DO2, VO2, and temperature responses to incremental dobutamine infusion in newborn and growing sheep, a species which, like humans [16], has abundant BAT in the initial days after birth [10, 17, 18]. To determine if responses observed in newborn lambs were related to a predominant a1-, b1- or b2-adrenoceptor subtype or to an interaction between these subtypes, additional experiments were performed with individual or combined adrenoceptor blockade.
Materials and methods The study was performed in 35 Border-Leicester cross lambs aged 1±2 days (n = 20), 7±10 days (n = 7), and 6±8 weeks (n = 8). All experiments were undertaken in accordance with the guidelines of the National Health and Medical Research Council of Australia
and were approved by the Monash University Animal Experimentation Committee. Lambs were surgically prepared as previously described [19]. Briefly, anaesthesia was induced with i.m. ketamine (5 mg/kg) and xylazine (0.1 mg/kg), followed by i. v. a-chloralose (15±25 mg/kg). A stable plane of surgical anaesthesia, assessed by the absence of canthal reflexes and presence of stable haemodynamics, was then maintained in all age groups with a continuous i. v. infusion of achloralose (10±25 mg/kg per hour). Animals were intubated with an endotracheal tube and ventilated with oxygen-enriched air, using volume-cycled ventilation (607 animal respirator, Harvard Apparatus, USA), a tidal volume of 10 ml/kg, and PEEP of 4±5 cmH2O. Blood gases were performed at frequent intervals and ventilation was adjusted to maintain arterial pH > 7.35, arterial PO2 at 100±130 mmHg, and arterial PCO2 at 35±40 mmHg, while base deficits > 2 mmol/l were corrected with sodium bicarbonate. The neck was incised in the midline and polyvinyl catheters were advanced through the left external jugular vein to the superior vena cava for administration of drugs and maintenance fluid therapy (2:1 mixture of crystalloid and colloid infused at 15 ml/kg per hour). After a left thoracotomy was performed in the fourth intercostal space, the adjacent ribs were sectioned anteriorly and posteriorly, the pericardium was incised over the pulmonary trunk and, in 1±2 day-old lambs, the ductus arteriosus was ligated. Cannulae were inserted through adventitial purse-string sutures in the descending thoracic aorta, pulmonary trunk, and left atrial appendage for pressure measurement and/or blood sampling. An ultrasonic, perivascular flow probe (10±14 mm diameter, Transonics Systems, USA) was placed around the ascending aorta. Central temperature was measured in 24 animals with a Swan-Ganz catheter inserted into the pulmonary trunk, while rectal temperature was measured in the remaining 11 animals. Pilot studies in our laboratory indicated that haemodynamic and blood gas variables remained stable for at least 2 h following completion of surgery in this type of experimental preparation. The main study was performed in lambs aged 1±2 days (n = 7, weight 5.0 0.4 kg, mean SEM), 7±10 days (n = 7, 7.0 0.6 kg), and 6±8 weeks (n = 8, 15.3 0.7 kg). After completion of surgery, blood pressure and cardiac output were monitored for 15±30 min to ensure that the experimental preparation was haemodynamically stable (i.e., variation < 10 %). Subsequently, baseline haemodynamics were recorded and blood samples were withdrawn from the aortic and pulmonary arterial catheters for blood gas analysis. Dobutamine (David Bull Laboratories, Australia) was then infused intravenously at incremental rates of 1, 2.5, 5, 7.5, 10, 15, 20, 30, and 40 mg/kg per minute. After attainment of steady-state haemodynamics, measurements were repeated 5±10 min into each dose and the infusion increased to the next dose. On the basis of results obtained from the main protocol, this infusion regimen was repeated in an additional four subgroups of 1±2 day-old lambs (n = 13, 4.8 0.2 kg) after pretreatment with either: 1) the a1adrenoreceptor antagonist prazosin (Sigma-Aldrich, Australia) [20], 0.2 mg/kg i. v. bolus then a continuous i. v. infusion of 0.2 mg/ kg per hour (n = 3); 2) the b1-adrenoceptor antagonist CGP 20712A (Ciba Geigy, Basel) [21], 50 mg/kg i. v. bolus followed by an infusion of 50 mg/kg per hour (n = 3); 3) the b2-adrenoceptor antagonist ICI 118551 (RBI, USA) [22], 0.2 mg/kg i. v. bolus followed by an infusion of 0.2 mg/kg per hour (n = 3); or 4) combined a1-, b1-, and b2-adrenoreceptor blockade via simultaneous infusion of prazosin, CGP 20712A, and ICI 118551 (n = 4), at the same doses as used in the individual blockade studies. At the end of the experiment, animals were killed with an overdose of pentobarbitone sodium. Aortic blood pressure was measured with a transducer (CDX111, COBE Laboratories, USA), referenced to atmospheric pres-
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Table 1 Haemodynamic variables during dobutamine infusion. Values are mean SE; n = 7, 7, and 8 for 1±2 day-old, 7±8 day-old, and 6±8 week-old lambs, respectively. P refers to analysis of variance of dose-response curve Dobutamine infusion rate (mg/kg/min) Variable
Age
0
2.5 a
174 7 211 10 215 14b 246 9 165 9 181 7
5
10
15
20
30
40
P
229 8 260 7 203 8
253 8 292 12 224 8
276 7 299 5 240 6
295 8 308 5 246 7
317 7 318 7 261 6
326 6 327 9 263 6
< 0.001 < 0.001 < 0.001
Heart rate (beats/min)
1±2 days 7±10 days 6±8 weeks
Mean aortic pressure (mmHg)
1±2 days 7±10 days 6±8 weeks
62 4 68 5 79 5
62 4 66 4 79 4
61 4 67 5 78 3
57 5 65 3 75 2
56 5 66 4 73 2
54 5 63 4 72 2
53 5 60 4 67 2
54 5 57 3 65 2
< 0.025 0.001 < 0.001
Left atrial pressure (mmHg)
1±2 days 7±10 days 6±8 weeks
5.4 0.9 5.7 1.0 3.8 0.7
3.8 0.7 4.4 0.9 2.6 0.6
3.9 0.6 4.2 0.8 2.2 0.6
4.4 0.8 4.5 1.0 2.8 0.5
4.9 0.8 4.5 0.8 2.7 0.6
5.3 0.9 5.4 1.0 4.1 1.0
5.8 0.9 6.1 1.0 3.8 0.6
6.5 0.8 7.3 1.1 4.6 0.8
< 0.001 < 0.001 0.002
Cardiac index (ml/min per kilogram)
1±2 days 7±10 days 6±8 weeks
162 11 183 12 165 18b 198 18 96 8 103 7
208 16 205 17 111 9
249 15 236 23 135 9
266 14 236 18 147 13
275 12 245 18 164 13
295 15 260 16 170 12
309 19 275 16 178 11
< 0.001 < 0.001 < 0.001
a
P < 0.025, 1±2 day-old vs 7±10 day-old lambs P < 0.005, 7±10 day-old vs 6±8 week-old lambs
b
sure at the level of the midthoracic vertebral spine. Cardiac output was obtained by measuring ascending aortic blood flow with an ultrasonic flowmeter (T208, Transonic Systems, USA). Outputs from pressure and flow transducers were amplified using an 8-channel programmable signal conditioner (Cyberamp 380, Axon Instruments, USA) and displayed continuously on a direct-writing recorder (Neotrace 800Z, Neomedix Systems, Australia). Pressure and flow signals were digitized at a sampling rate of 500 Hz for 20 s, and the data stored on computer for subsequent offline analysis. Blood gases were analyzed at the measured body temperature with a blood gas analyser (ABL 500, Radiometer, Denmark). Haemoglobin (Hb) and haemoglobin O2 saturation (HbS) were measured with a hemoximeter (OSM2, Radiometer, Denmark). Oxygen content (ml/dl) in the aorta (CAOO2) and pulmonary artery (CPAO2) were calculated as (1.36HbHbS/100) + (0.003PO2). The systemic arteriovenous (A-V) O2 content difference was calculated as CAOO2±CPAO2 and the systemic O2 extraction coefficient (ERO2) as (CAOO2±CPAO2) / CAOO2, while DO2 was derived from the product of cardiac output and CAOO2, and VO2 from the product of cardiac output and (CAOO2±CPAO2). Results are expressed as mean SE and were analyzed using Statistical Package for the Social Sciences Version 9.0.1 (SPSS, USA). Dobutamine dose-response curves were analyzed with a mixed factorial ANOVA, while baseline variables in the three age groups were compared with one-way ANOVA. The relationship between VO2 and DO2, and between VO2 and body temperature were analyzed with least squares linear regression. Significance was accepted at the P < 0.05 level.
Results Dobutamine increased heart rate and cardiac index, lowered mean aortic blood pressure, and produced a biphasic response in left atrial pressure in all age groups (P < 0.025±0.001; Table 1). During dobutamine infusion, CAOO2 fell in 1±2 and 7±8 day-old lambs (P < 0.001) but increased in 6±8 week-old lambs (P = 0.001), while CPAO2 decreased substantially in 1±2 day-old lambs (P < 0.001) and rose in 7±8 day-old
and 6±8 week-old lambs (P < 0.001). In association with these changes, the A-V O2 content difference and ERO2 increased in 1±2 day-old lambs (P < 0.001), but fell in 7±8 day-old and 6±8 week-old lambs (P£0.001; Table 2). DO2 increased progressively in the three groups (P < 0.001), with no difference in responses after allowing for age-related baseline differences (P > 0.3; Fig. 1A). Dobutamine also augmented VO2 in all ages, in the absence of any observable muscle shivering. However, this rise in VO2 was most pronounced in 1±2 day-old lambs, with the increase above baseline at infusion rates ³10 mg/kg per minute (8.2 1.7 ml/min per kilogram) being sevenfold to 12-fold greater than the increments of 0.7 0.7 ml/min per kilogram and 1.1 0.3 ml/min per kilogram observed in the 7±10 dayold and 6±8 week-old groups, respectively (Fig. 1B). Moreover, the slope of the relationship between changes in VO2 and DO2 in 1±2 day-old lambs (0.91 0.14, r = 0.92) exceeded that of 7±10 days (0.34 0.08, r = 0.84, P < 0.005) and 6±8 week-old lambs (0.15 0.03, r = 0.91, P < 0.001). In association with increases in VO2, body temperature in 1±2 day-old animals rose by 1.3 0.5 C (P < 0.001), but did not change significantly in 7±10 day-old or 6±8 week-old lambs (Fig. 1C). In addition, increases in body temperature and VO2 were linearly related (r = 0.99), with VO2 increasing 3.7 0.3 ml/min per kilogram per C rise in body temperature (P < 0.001). Moreover, increases in both cardiac output and O2 extraction contributed to rises in VO2 in 1±2 day-old lambs (Fig. 2), but at infusion rates ³10 mg/kg per minute, the rise in cardiac output (71 9 %) exceeded that of O2 extraction (32 9 %, P < 0.001). With the exception of a » 10 % fall in DO2 after b1 blockade (P < 0.025) and a trend to reductions in DO2
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Table 2 Blood gas variables during dobutamine infusion. Values are mean SE; n = 7, 7, and 8 for 1±2 day-old, 7±8 day-old, and 6±8 week-old lambs, respectively. P refers to analysis of variance of dose-response curve Dobutamine infusion rate (mg/kg/min) Variable
Age
0
2.5
5
10
15
20
30
40
P
b
CAOO2 (ml/dl)
1±2 days 7±10 days 6±8 weeks
16.4 0.8 12.2 0.9 13.4 0.5
16.3 0.8 11.7 0.9 13.9 0.6
15.9 0.6 12.0 1.0 14.3 0.6
15.3 0.8 11.6 1.0 14.4 0.5
14.8 0.5 11.9 0.9 14.5 0.5
14.4 0.6 11.4 1.0 14.2 0.4
13.8 0.6 11.3 1.1 13.9 0.5
13.4 0.5 10.8 1.0 13.4 0.5
< 0.001 < 0.001 0.001
CPAO2 (ml/dl)
1±2 days 7±10 days 6±8 weeks
12.2 0.9b 7.7 0.9 8.9 0.6
12.7 0.9 8.4 1.0 9.9 0.7
12.3 0.6 8.6 1.0 10.4 0.7
10.7 0.3 8.5 0.9 10.5 0.5
9.5 0.4 8.6 1.0 10.5 0.7
8.7 0.4 8.2 1.1 10.6 0.5
7.7 0.3 7.8 0.5 10.7 0.5
7.4 0.3 10.4 0.5 10.4 0.5
< 0.001 < 0.001 < 0.001
A-V O2 Content difference (ml/dl) ERO2
1±2 days 7±10 days 6±8 weeks
4.2 0.4 4.6 0.4 4.5 0.4
3.6 0.3 3.3 0.3 4.0 0.3
3.6 0.3 3.4 0.2 3.9 0.4
4.6 0.8 3.1 0.4 3.8 0.3
5.3 0.6 3.4 0.3 4.0 0.4
5.6 0.6 3.2 0.3 3.5 0.2
6.1 0.5 3.5 0.4 3.2 0.2
6.0 0.3 3.4 0.5 3.0 0.2
< 0.001 < 0.001 < 0.001
1±2 days 7±10 days 6±8 weeks
0.26 0.03a 0.22 0.02 0.23 0.02 0.29 0.03 0.35 0.03 0.39 0.03 0.44 0.02 0.45 0.01 < 0.001 0.38 0.03 0.29 0.03 0.29 0.03 0.27 0.03 0.29 0.03 0.29 0.03 0.33 0.04 0.34 0.06 0.001 0.34 0.03 0.29 0.03 0.28 0.03 0.27 0.02 0.28 0.03 0.25 0.02 0.23 0.01 0.23 0.01 < 0.001
a
P < 0.05 P < 0.005, 1±2 vs 7±10 day-old lambs
b
(P = 0.06) and VO2 (P = 0.07) after combined a1, b1, and b2 blockade, adrenoceptor blockade did not alter resting DO2, VO2 or body temperature in 1±2 day-old lambs. However, while individual adrenoceptor blockade did not significantly attenuate thermogenic responses, pretreatment with combined a1, b1, and b2 blockade prevented rises in DO2 (P > 0.2; Fig. 3A), VO2 (P = 0.2; Fig. 3B), and body temperature (P > 0.9; Fig. 3C).
Discussion This study has produced three main findings. First, dobutamine had a pronounced thermogenic effect in newborn lambs, but this disappeared by the end of the first postnatal week. Second, the marked rise in VO2 which accompanied this thermogenic action utilized most of the dobutamine-induced increase in DO2. Third, increases in body temperature and VO2 in newborn lambs were not substantially affected by individual a1, b1 or b2 adrenoceptor blockade, but were blunted by combined blockade of these adrenoceptors. Thermogenic responses in the newborn mammals occur via two types of processes, namely shivering in skeletal muscle and NST in BAT [23]. The dose-dependent rises in body temperature and VO2 in the absence of any muscle shivering observed with infusion of dobutamine in 1±2 day-old animals in our study, as well as the close relationship between changes in VO2 and body temperature, was therefore quite consistent with the notion that this catecholamine, like the endogenous stimulant noradrenaline [10, 11], activated NST in the newborn. In contrast to 1±2 day-old lambs, body temperature was unchanged and increases in VO2 during dobutamine infusion were substantially lower by the
end of the first week after birth, indicating that the capacity for such thermogenesis was confined to a narrow window within the initial days after birth. This pattern is in accord with the developmental characteristics in sheep of the two elements involved in NST, namely BAT and uncoupling protein. Specifically, while BAT is the principal constituent of body fat deposits at birth [18], a relatively rapid transformation to white adipose tissue subsequently ensues [17, 18, 24] in association with a disappearance of uncoupling protein mRNA from adipocytes in the first few days after birth [24, 25] and a marked fall in uncoupling protein content in adipocytes by the end of the first postnatal week [25]. It is likely, therefore, that, as in the adult, rises in VO2 evident in 7±10 days and 6±8 week-old lambs were mainly related to an increase in tissue metabolism occurring secondary to factors such as the stimulation of substrate mobilization and intermediary metabolism [2, 6], and to rises in O2 usage of specific organs such as the heart and kidney [26]. The resting levels of cardiac output, DO2, and VO2 in our study showed the expected decline with advancing postnatal age [27]. However, even though rises in DO2 produced by dobutamine were similar at the three ages studied, regression analysis indicated that the dramatic increase in VO2 in 1±2 day-old lambs had major consequences for the balance between DO2 and VO2 because rises in VO2 in 1±2 day-old lambs utilized » 90 % of accompanying elevations in DO2, compared to only 34 % in 7±10 day-old lambs and 15 % in 6±8 weekold lambs. Furthermore, the substantial rises in VO2 observed in 1±2 day-old lambs during dobutamine infusion were supported not only by an elevation in cardiac index, but also by an appreciable increment in ERO2, which is indicative of a utilization of systemic O2 extrac-
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Fig. 2 Relationship between cardiac index and systemic arteriovenous (A-V) O2 content difference, expressed as a percentage of baseline, during dobutamine infusion in 1±2 day-old (*), 7±10 day-old (&), and 6±8 week-old lambs (~). Note that all points lie above the line of identity, indicating that changes in cardiac index exceeded those of A-V O2 content difference
Fig. 1 Effect of dobutamine infusion on A systemic O2 delivery, B systemic O2 consumption, and C body temperature in 1±2 day-old (*), 7±10 day-old (&), and 6±8 week-old lambs (~). ** P < 0.005 and *** P < 0.001 from analysis of variance of dose-response curve. Note that resting systemic O2 delivery in 7±10 day-old lambs was lower than in 1±2 day-old lambs (P < 0.025) but higher than in 6±8 week-old lambs (P < 0.01), while systemic O2 consumption in 1±2 and 7±10 day-old lambs exceeded that of 6±8 week-old animals (P < 0.005)
tion reserves [28]. On the basis of present results, however, we are unable to determine whether this increase in ERO2 resulted from a striking capacity for O2 removal by activated BAT [12, 29], a rise in O2 extraction by other systemic tissues because of a diversion of cardiac output to meet the very high flow requirements of activated BAT [12, 23], or a combination of these two factors. Thermogenic stimulation of isolated brown adipocytes derived from lambs has been reported to occur mainly via b1-adrenoceptors and to a lesser extent via a1-adrenoceptors [30]. However, in our study, body temperature and VO2 responses were not altered significantly with individual a1-, b1-, or b2-adrenoceptor blockade suggesting that, in the intact animal, the thermogenic response was not due to a predominant effect of dobutamine on one of these adrenoceptors. In contrast, dobutamine-induced thermogenesis and rises in VO2 were markedly attenuated with combined a1-, b1-, and b2-adrenoceptor blockade, implying that thermogenic activation occurred via an interaction between two or more of these adrenoceptor subtypes. Consistent with this proposition, a1- and b1-adrenoceptors are known to interact in vivo, with a1-adrenoceptors having a significant potentiating effect on b1-adrenoceptor-induced responses [31]. However, because increases in VO2 during non-shivering thermogenesis may also be dependent on rises in BAT blood flow [32], we cannot exclude the possibility that the blunting of rises in VO2 and body temperature evident after combined adrenoceptor blockade
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Fig. 3 Changes in A systemic O2 delivery, B systemic O2 consumption, and C body temperature during dobutamine infusion in 1±2 day-old lambs before (*) or after pre-treatment with a1 (&), b1 (!) or b2-adrenoceptor blocker (^), or combined a1-, b1-, and b2-adrenoceptor blockade (*)
was also in part related to the associated dampening of increases in DO2 (Fig. 3A). An additional possibility, raised by findings in isolated adult rat brown adipocytes, was that dobutamine also stimulated lipolysis via a b3- [13, 15] or an atypical non-b1,2,3-adrenoceptor [14]. We cannot entirely exclude a role for these adrenoceptor subtypes in the thermogenic response observed in our study. However, in accord with the considerable species variation apparent in the role of b3-adrenoceptors in BAT-related thermogenesis [33, 34, 35] and the limited role that this receptor subtype plays in the induction of uncoupling protein mRNA in lambs [36], preliminary information suggests that stimulation of b3-adrenoceptors has no significant effect on the thermogenic activity of BAT or on VO2 in newborn lambs [37]. Furthermore, a major role for an atypical non-b1,2,3-adrenoceptor seems unlikely given the profound effect of combined a1-, b1-, and b2-adrenoceptor blockade in newborn lambs (Fig. 3). Our study had four main potential limitations. First, experiments were performed under general anaesthesia, in large part because catecholamine infusion into the conscious newborn may result in arousal, with the confounding effect of rises in VO2 due to the associated increase in muscle activity [38]. As general anaesthesia may have altered not only resting variables but also the magnitude of responses in DO2, VO2, and body temperature during dobutamine infusion, it remains unclear to what extent the findings of the present study can be extrapolated to the conscious newborn. Second, because a shared variable (cardiac output) was used in the calculation of DO2 and VO2, comparison of changes between these variables may have been influenced by the presence of mathematical coupling [39]. It is likely that the effect of such coupling was very minor, however, due not only to the accuracy ( 2 %) of blood flow measurements provided by ultrasonic flow technology, but also because the variation of DO2 over a wide range during dobutamine infusion minimized the effects of random measurement errors [26, 40]. Third, the use of the same sequence of incremental infusion rates of dobutamine rather than a randomized protocol may have resulted in tissue accumulation of dobutamine. Whilst we cannot exclude such an occurrence, it was unlikely that drug accumulation underlay the dramatic differences in VO2, as other responses (e.g., cardiac index and DO2) were similar in the three age groups. Finally, the short period of each infusion rate (5±10 min) may not have permitted
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the attainment of true steady-state metabolic conditions. However, this was an avoidable accompaniment of the number of dobutamine doses employed in our protocol because of the risk that prolonged catecholamine infusion in the newborn (i.e., ³2 h) may have adversely affected cardiac function and cardiomyocyte ultrastructure [41]. Dobutamine is widely used as an inotropic agent in critically ill neonates [42]. BAT is present in the human fetus from around the 20th week of gestation onwards and is abundant in both the pre-term and full-term neonate [16]. Uncoupling protein is also detectable at birth in preterm and term infants [43]. Furthermore, noradrenaline infusion causes large increases in VO2 in newborn infants [38]. Taken together, these observations raise the possibility that a thermogenic response to dobutamine, and perhaps other adrenergic inotropes, may also occur in the human neonate, particularly with high-dose infusion regimens. In view of the dramatic
findings of our study, this question warrants specific examination in neonates undergoing inotropic therapy, because if dobutamine-induced increases in cardiac output and DO2 are diverted towards activated BAT to meet its very high blood flow and O2 requirements, then this may potentially limit increases in blood flow and O2 delivery to other systemic organs and thus predispose to the development of tissue hypoxia. The accompaniment of thermogenic responses by increased systemic O2 extraction due to large falls in pulmonary O2 arterial content (Table 2) suggests that blood gas analysis of mixed-venous blood samples [7, 8, 9] could provide a relatively simple means of detecting inotrope-induced thermogenesis in neonates. Acknowledgements We thank Karyn Forster for her technical assistance in carrying out the experiments of this study as well as Kellie Eede and Ann Oates for their help in preparation of this manuscript. We also thank Ciba-Geigy for the donation of CGP 20712A.
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