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Effects of temperature on lung and blood gases in the South American rattlesnake Crotalus durissus terrificus
Comparative Biochemistry and Physiology Part A 121 (1998) 7 – 11
Effects of temperature on lung and blood gases in the South American rattlesnake Crotalus durissus terrificus Tobias Wang a,*, Augusto S. Abe a, Mogens L. Glass b b
a Department of Zoology, UNESP-Rio Claro, 13500 Rio Claro SP, Brazil Department of Physiology, School of Medicine, Uni6ersity of Sao Paulo, 14049 Riberao Preto SP, Brazil
Received 25 February 1998; received in revised form 3 May 1998; accepted 1 June 1998
Abstract The effects of temperature on lung and blood gases were measured in the South American rattlesnake (Crotalus durissus terrificus). Arterial blood and lung gas samples were obtained from chronically cannulated animals at 15, 25, and 35°C. As expected for reptiles, arterial pH fell with increased temperature (0.018 U °C − 1 between 15 and 25°C and 0.011 U °C − 1 between 25 and 35°C) while lung gas PCO2 rose from 5.8 mmHg at 15°C to 13.2 mmHg at 35°C. Concurrently, lung gas PO2 declined from 132 mmHg at 15°C to 120 mmHg at 35°C, and arterial PO2 increased from 33 to 76 mmHg in that temperature range. Arterial haemoglobin O2 saturation rose from 0.53 at 15°C to 0.83 at 25°C but became slightly reduced (0.77) with a further elevation of temperature to 35°C. Arterial haemoglobin concentration increased from 1.96 to 2.53 mM between 15 and 35°C, consistent with higher demands on oxygen delivery to tissues at elevated temperatures. Moreover, the substantial increase of haemoglobin O2 saturation between 15 and 25°C conforms to the idea that reduction of the central vascular right-to-left shunt (pulmonary bypass of systemic venous return) is associated with high metabolic demands. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Reptile; Snake; Crotalus; Temperature; Acid – base balance; O2 transport; Lung gases; Arterial blood gases
1. Introduction Many ectothermic vertebrates undergo large diurnal and seasonal fluctuations in body temperature with associated changes in metabolism and in cardio-respiratory function. The effects of body temperature on arterial blood gases have been described for a number of reptiles and amphibians and, as a general rule, increased temperature is associated with a reduction in arterial pH, an elevation of arterial PCO2, and a rise of arterial PO2 [7,30,33,39]. Wood [37,38] attributed the increase in arterial PO2 to a reduction of blood oxygen affinity with increased temperature in combination with an incomplete saturation of arterial blood. In most * Corresponding author. Present address: Center for Respiratory Adaptation, Institute of Biology, Odense University, 5230 Odense M, Denmark. Tel.: + 45 65 572460; fax: +45 65 930457; e-mail: [email protected]
amphibians and reptiles, the systemic and pulmonary circulations are not completely separated and the arterial blood is, therefore, a mixture of venous systemic blood and blood returning from the lungs (central right-to-left shunt; R–L shunt). As a consequence, the oxygen saturation of the arterial blood becomes reduced and arterial PO2 can be considered a dependent variable determined by the resulting haemoglobin O2 saturation and the blood oxygen affinity. Assuming that haemoglobin oxygen saturation (HbO2 saturation) is unaffected by temperature, PO2 increases with temperature due to right-shift of the oxygen dissociation curve [38]. Few studies report on the arterial blood oxygen saturation in relation to temperature in reptiles, but in the lizard Iguana iguana and the snake Pituophis melanoleucus, HbO2 saturation is constant and temperature-independent over a wide temperature range [27,31]. However, in turtles and varanid lizards, micro-
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T. Wang et al. / Comparati6e Biochemistry and Physiology, Part A 121 (1998) 7–11
sphere injections document that the R – L shunt is reduced with increased temperature [9,15]. A reduction in the R–L shunt is expected to increase arterial HbO2 saturation and would contribute to the increased PO2 with elevated temperature. In the present study we provide direct measurements of arterial HbO2 saturation, haemoglobin concentration, PO2, pH, as well as lung gas pressures at three different temperatures in the South American rattlesnake Crotalus durissus terrifus.
2. Materials and methods
2.1. Experimental animals Six rattlesnakes (400 – 800 g) were obtained from the Butantan Institute (Sao Paulo, Brazil) and kept in outdoor enclosures at the campus of Universidade Estadual Paulista in Rio Claro (Sao Paulo, Brazil) until experimentation. The snakes were fed mice until 7–10 days before surgery.
2.2. Surgery Each snake was anaesthetised by inhalation of ether vapour until ciliary reflexes were absent and it ceased to breathe spontaneously. Then the trachea was intubated using a small rubber tube and the animals were ventilated manually every 2 – 3 min with a mixture of air and ether vapour to maintain anaesthesia. The coeliac artery was occlusively cannulated by advancing a PE50 catheter containing heparinized saline into the dorsal aorta. In addition, a pulmonary catheter (PE 160 with a flared end and sideholes) was placed with the tip protruding 0.2 – 0.5 cm into the lumen of the vascularized portion of the lung. Subsequently, lung parenchyma was tied around the catheter, which was then fed out dorsally and secured to the back of the snake with a few sutures. Both incisions were closed with intermittent sutures and the snake was allowed to recover from the anaesthesia for 18–36 h at 25°C.
2.3. Analysis of blood and lung gases Arterial PO2 and pH were measured using a Radiometer (Copenhagen, Denmark) Blood Micro System that maintained the electrodes at the experimental temperature. Both electrodes were calibrated immediately prior to measurements. For the PO2 measurements an experimentally determined liquid/gas correction factor as well as the effects of blood metabolism were taken into account. Total oxygen content of the arterial blood ([O2]a) was measured as described by Tucker [32] taking the correc-
tion by Bridges et al. [1] into account. Haemoglobin concentration ([Hb]) was measured spectrophotometrically at 540 nm following conversion to cyanomethhaemoglobin and applying a millimolar extinction coefficient of 11.0 [40]. Because some previous studies on reptiles have reported high concentrations of nonfunctional haemoglobin [21], we obtained an in vitro relationship between the oxygen carrying capacity of the haemoglobin (HbO2cap) and the haemoglobin concentration ([Hb]) for each snake. To this purpose, a 1.5 ml blood sample was equilibrated in a rotating tonometer (Eschweiler, Germany) with a gas mixture of 30% O2 and 2% CO2 (balance N2) for 30 min, after which total oxygen content and [Hb] were determined as described above. From the measurement of O2 content, HbO2cap was calculated by subtracting dissolved O2 (aO2 · PO2, where aO2 is the human O2 solubility coefficient [2]). Having established the correlation between HbO2cap and [Hb], the arterial oxygen haemoglobin saturation (arterial HbO2 sat) was calculated as arterial HbO2 sat= 100 · [(CaO2 − (aO2 · PaO2))/ HbO2cap)]%. Lung gas samples of 5 ml were drawn into gas tight syringes and analysed immediately for O2 and CO2 concentrations by means of a Beckman Medical Infrared CO2 Analyzer (LB-2) and an Applied Electrochemistry Oxygen Analyzer (S-3A/L) connected in series. The analysers were calibrated with high precision gas mixtures from Aga (Brazil).
2.4. Experimental protocol Experiments were postponed for a minimum of 18 h after surgery. The snakes were maintained in a climatic chamber (Favolet, Sao Paulo, Brazil) that provided both visual and acoustical isolation of the animals throughout the measurements. Measurements were conducted at three different temperatures (15, 25, and 35°C). The experiments always started at 25°C, while the second experimental temperature was chosen at random and no measurements were taken within a 12 h period following a change of temperature. For each snake at each temperature, at least 20 lung gas and three arterial blood samples were obtained and analysed. The measurements of blood gases were interspersed by approximately 2 h, while the lung gas measurements were taken at approximately 15 min intervals. For each snake, a mean value of blood and lung gases was calculated on the basis of all the measurements at the respective temperatures. For the analysis of pH, [Hb], [O2]a, and PO2, a blood sample of 600 ml was withdrawn, but since some of the blood could be re-injected after analysis, each analysis was only associated with a blood loss of approximately 250 ml.
T. Wang et al. / Comparati6e Biochemistry and Physiology, Part A 121 (1998) 7–11
2.5. Statistics A one-way ANOVA for repeated measures was employed to assess significant effects of temperature on the reported parameters. Differences among means were subsequently assessed using a Student-NewmanKeul’s test. A fiducial limit for significance of P 5 0.05 was applied and all data in the text and figures are presented as mean9 1 S.D.
3. Results Arterial pH decreased significantly by 0.0189 0.007 U °C − 1 between 15 and 25°C and by 0.01190.003 U °C − 1 between 25 and 35°C (Fig. 1A). This reduction in pHa reflects a significant increase in intrapulmonary PCO2 from 5.8 9 1.7 mmHg at 15°C to 13.2 9 2.2 mmHg at 35°C (Fig. 1B). Intrapulmonary PCO2 increased relatively more within the low temperature range compared to the high range; D log(PCO2)/DT was 0.23 between 15 and 25°C and only 0.12 between 25 and 35°C. Concurrent with the rise in PCO2, increased temperature was associated with a reduction in intrapulmonary PO2 that declined from 131.99 3.3 mmHg at 15°C to 119.5 95.7 mmHg at 35°C (Fig. 1C). Conversely, arterial PO2 rose from 32.795.8 mmHg at
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15°C to 66.79 3.4 at 25°C and reached 75.794.4 mmHg at 35°C (Fig. 1C). Based on the alveolar gas equation [20], effective ventilation (V: eff) relative to pulmonary CO2 output (V: CO2) was calculated as V: eff/V: CO2 = RT/PLCO2, Where PLCO2 is the PCO2 in the gas exchange zone of the lung, R is the gas constant, and T is the absolute temperature (K). Based on the mean values for PLCO2 obtained on Crotalus in the present study, V: eff/V: CO2 is predicted to decrease from 138 at 15°C to 65 at 35°C (Fig. 1D). Arterial HbO2 saturation increased significantly from 0.539 0.09 at 15°C to 0.83 9 0.03 at 25°C, while a further increase in temperature to 35°C was associated with a non-significant reduction in HbO2 saturation to 0.7790.08 (Fig. 1E). Also, the haemoglobin concentration of the arterial blood increased significantly with rising temperature from 1.969 0.15 mM at 15°C to 2.5390.20 mM at 35°C (Fig. 1F). The total oxygen carrying capacity of the blood determined in vitro increased correspondingly. For all snakes, the determinations of oxygen carrying capacity of the haemoglobin (HbO2cap) and the haemoglobin concentration ([Hb]) were within 9 3% of each other, indicating that virtually all haemoglobin was functional, i.e. that methhaemoglobin levels were insignificant.
4. Discussion
4.1. Effects of temperature on arterial pH The negative DpH/DT in Crotalus is consistent with data for most ectothermic vertebrates [10,33] and with reports for other species of snakes [3,19,27,30]. Reeves [24] suggested that the change in pH parallels that of pK for the alpha imidazole system such that the degree of protein ionization is maintained constant (alphastat regulation). In many species, the DpH/DT is less than required by the hypothesis [7,10,30]. In Crotalus, the pH change between 15 and 25°C was compatible with alphastat regulation, whereas the change was smaller than predicted between 25 and 35°C.
4.2. Effects of temperature on blood and lung gases
Fig. 1. The effects of temperature on lung and blood gases in the South American rattlesnake (Crotalus durissus terrificus). A, Arterial pH; B, Intrapulmonary PCO2 (PLCO2); C, Arterial PO2 and intrapulmonary PO2 (PLO2); D, Effective ventilation (Veff) relative to pulmonary CO2 output (V: CO2); E, Arterial haemoglobin oxygen saturation (HbO2 saturation); F, Arterial haemoglobin concentration ([Hb]). Mean values 9 1 S.D. (N =6).
The values for PO2 and PCO2 in the lung gas of Crotalus are consistent with direct measurements of lung gases from the viper Vipera anthina palestinae [8] and end-expired gases in the python Morelia spilotes [6]. In these species the PLO2 ranged from 120 to 145 mmHg and PLCO2 from 5 to 20 mmHg. The data for Vipera and Morelia were confined to the narrow temperature range of 22–26°C and the effects of tempera-
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ture were, therefore, not evaluated. In Crotalus a marked decrease of PLO2 from 132 mmHg (15°C) to 120 mmHg (35°C) was accompanied by increases of PaO2, whereby the PLO2 −PaO2 difference was reduced from about 100 mmHg to about 45 mmHg. Also, the increase of PaO2 with temperature in Crotalus (Fig. 1) is consistent with previous reports on snakes and represents a general pattern among reptiles and amphibians [7,19,27,29]. Wood [37,38] explained this pattern as a consequence of the R – L shunt. In its presence, arterial HbO2 saturation is reduced due to the admixture of O2 poor systemic blood to the O2 rich blood returning from the lungs, and PaO2 becomes a dependent variable determined by the resulting haemoglobin oxygen saturation and the blood oxygen affinity. Thus, as increased temperature lowers blood oxygen affinity, PaO2 will increase even if haemoglobin oxygen saturation is constant. In Crotalus, however, the arterial HbO2 saturation increased markedly between 15 and 25°C and thus contributes to the pronounced elevation of PaO2 within this temperature range. In contrast to the large increase in HbO2 saturation observed in Crotalus, two previous studies on snakes and iguanas report a temperature-independent HbO2 saturation over a wide temperature range [27,31]. In reptiles, the PLO2 −PaO2 difference is the combined result of diffusive resistance of the barrier separating lung gas and blood, ventilation-perfusion inhomogeneity within the lung and intrapulmonary and central vascular R– L shunts [36]. Since a change in some or all of these factors will alter arterial HbO2 saturation, it is difficult to establish the mechanism behind the large increase of HbO2 saturation between 15 and 25°C in the present study. In sea snakes, an intrapulmonary shunt of almost 20% has been determined [25]. Similar values have been reported for turtles, tegu lizards, and alligators [12,14,22,26], while values for monitor lizards are substantially smaller [13]. In turtles, the pulmonary shunt flow increases whenever pulmonary blood flow increases at constant temperature [14], but no studies have addressed this aspect in relation to temperature. Because the ophidian lung consists of an anterior, highly vascularized section and a posterior, virtually non-vascularized region [18], ventilation-perfusion inhomogeneity and stratification also increase the PLO2 − PaO2 difference [5,6,8,23]. However, these factors probably contribute little to the PLO2 − PaO2 difference in resting animals [13,14]. Therefore, it seems reasonable to suggest that a reduction in cardiac R–L shunt with increased temperature in Crotalus can explain most of the increased HbO2 saturation between 15 and 25°C. Consistent with this view, a decrease of the cardiac R – L shunt with rising temperature has been reported for the turtle Chrysemys picta bellii [9] and the monitor lizard Varanus niloticus [15]. This finding is compatible with the hypothesis that
reptiles reduce the magnitude of R–L shunt during conditions of elevated metabolic rate, whether due to exercise, digestion, or increased temperature [11,35]. In addition to reduction in R–L shunt, the large increase in blood [Hb] and, consequently, in blood oxygen carrying capacity, of Crotalus promotes oxygen delivery at higher temperatures.
4.3. A possible conflict between acid–base balance and O2 transport at high temperatures The control of pulmonary ventilation performs the dual role of regulating arterial acid–base balance through adjustments of PCO2 and of regulating arterial oxygen levels. Potentially, a conflict between these functions may develop in amphibians and reptiles exposed to high body temperature. The present and most previous studies show that arterial pH decreases with increased body temperature [30]. This negative DpH/DT is achieved by ventilatory adjustments to elevate PaCO2, although changes in bicarbonate levels may contribute [29,33]. The increased PaCO2 is accomplished through a progressive decrease in effective ventilation relative to metabolic rate as temperature increases [3,4,16,19,27]. As a consequence of the progressive hypoventilation with increased temperature, intrapulmonary PO2 is progressively reduced (Fig. 1, [28]). This decline of PLO2 coincides with a reduction in blood oxygen affinity and must at some point reduce the HbO2 saturation of the blood leaving the lung. On this basis and using the two-compartment model, it has been predicted that there exists a critical temperature above which arterial oxygen levels (PO2 and HbO2 saturation) decrease due to impaired oxygen loading at the lungs [39]. Some experimental data support the existence of such a ‘breaking point.’ In the snake Coluber constrictor, arterial O2 content decreased upon exposure to body temperatures above 30°C [28]. Likewise, in the turtle Pseudemys floridana, both pulmonary venous and systemic arterial HbO2 saturation decreased with rising temperature [17]. The blood oxygen affinity of Crotalus is very low (P50 is approximately 50 mmHg at 30°C, pH 7.42 [34]). Accordingly, this species should be susceptible to this conflict. However, the high PLO2 (115 mmHg) at 35°C undoubtedly contributes to maintaining adequate arterial O2 levels. Moreover, a direct evaluation of impaired blood oxygen binding at high temperatures would require direct measurements of HbO2 saturation of the pulmonary venous blood. In conclusion, PaO2 increased with rising temperature, whereas lung gas PO2 decreased but still remained high. HbO2 saturation increased markedly between 15 and 25°C, suggesting a reduction in the cardiac R–L shunt. There was no unequivocal evidence for a conflict between oxygen transport and acid–base regulation in Crotalus exposed to high temperature. Clear signs of a
T. Wang et al. / Comparati6e Biochemistry and Physiology, Part A 121 (1998) 7–11
conflict may require more strenuous conditions such as a high metabolic rate during exercise or digestion, or during hypoxia.
Acknowledgements This study was supported by an equipment grant from FAPESP (89/2857-5) and CNPq (300603/91-6). T. Wang received support from the Danish Research Council.
References [1] Bridges CR, Bicudo JEPW, Lykkeboe G. Oxygen content measurements in blood containing haemocyanin. Comp Biochem Physiol 1979;62A:457–62. [2] Christoforides C, Hedley-Whyte J. Effect of temperature and hemoglobin concentration on solubility of O2 in blood. J Appl Physiol 1969;27:592 –6. [3] Dean JB, Gratz RK. The effect of body temperature and CO2 breathing on ventilation and acid–base status in the northern water snake Nerodia sipedon. Physiol Zool 1983;56:290–301. [4] Dmi’el R. Effect of activity and temperature on metabolism and water loss in snakes. Am J Physiol 1972;223:510–6. [5] Donnelly PM, Woolcock AJ. Ventilation and gas exchange in the carpet python, Morelia spilotes 6ariegata. J Comp Physiol B 1977;122:403 – 18. [6] Donnelly PM, Woolcock AJ. Stratification of inspired air in the elongated lungs of the carpet python, Morelia spilotes 6ariegata. Respir Physiol 1978;35:301–15. [7] Glass ML, Boutilier RG, Heisler N. Effects of body temperature on respiration, blood gases and acid–base status in the turtle Chrysemys picta bellii. J Exp Biol 1985;114:37–51. [8] Gratz RK, Ar A, Geiser J. Gas tension profile of the lung of the viper, Vipera xanthina palestinae. Respir Physiol 1981;44:165 – 76. [9] Heisler N, Glass ML. Mechanisms and regulation of central vascular shunts in reptiles. In: Johansen K, Burggren WW, editors. Cardiovascular Shunts. Phylogenetic, Ontogenetic and Clinical Aspects. Copenhagen: Munksgaard, 1985:334–53. [10] Heisler N. Comparative aspects of acid–base regulation. In: Heisler N, editor. Acid–Base Regulation in Animals. Amsterdam: Elsevier, 1986:397–450. [11] Hicks JW, Wang T. Functional role of cardiac shunts in reptiles. J Exp Zool 1996;275:204–16. [12] Hlastala MP, Standaert TA, Pierson DJ, Luchtel DL. The matching of ventilation and perfusion in the lung of the Tegu lizard, Tupinambis nigropunctatus. Respir Physiol 1985;60:277 – 94. [13] Hopkins SR, Hicks JW, Cooper TK, Powell FL. Ventilation and pulmonary gas exchange during exercise in the savannah monitor lizard (Varanus exanthematicus). J Exp Biol 1995;198:1783– 9. [14] Hopkins SR, Wang T, Hicks JW. The effect of altering pulmonary blood flow on pulmonary gas exchange in the turtle Trachemys (Pseudemys) script. J Exp Biol 1996;199:2207– 14. [15] Ishimatsu A, Hicks JW, Heisler N. Analysis of intracardiac shunting in the lizard, Varanus niloticus: a new model based on blood oxygen levels and microsphere distribution. Respir Physiol 1988;71:83 – 100. [16] Jackson DC, Palmer SE, Meadow WL. The effects of temperature and carbon dioxide breathing on ventilation and acid – base .
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status of turtles. Respir Physiol 1974;20:131 – 46. [17] Kinney JL, Matsuura DT, White FN. Cardiorespiratory effects of temperature in the turtle, Pseudemys floridana. Respir Physiol 1977;31:309 – 25. [18] Luchtel DL, Kardong KV. Ultrastructure of the lung of the rattlesnake, Crotalus 6iridis oreganus. J Morphol 1981;169:29– 47. [19] Nolan WF, Frankel HM. Effects of temperature on ventilation and acid – base status in the black racer snake, Coluber constrictor. Comp Biochem Physiol 1982;73A:57 – 61. [20] Otis AB. Quantitative relationships in steady-state gas exchange. In: Fenn WO, Rahn H, editors. Handbook of Physiology. Washington: American Physiology Society, 1964:681 – 98. [21] Pough FH. Blood oxygen transport and delivery in reptiles. Am Zool 1980;20:173 – 85. [22] Powell FL, Gray AT. Ventilation-perfusion relationships in alligators. Respir Physiol 1989;78:83 – 94. [23] Read J, Donnelly P. Stratification of blood flow in the elongated lungs of the carpet python. J Appl Physiol 1972;32:842–6. [24] Reeves RB. An imidazole alphastat hypothesis for vertebrate acid – base regulation: tissue carbon dioxide content and body temperature in bullfrogs. Respir Physiol 1972;14:219 – 36. [25] Seymour RS. Gas tensions and blood distribution in sea snakes at surface pressure and at simulated depth. Physiol Zool 1978;51:388 – 407. [26] Seymour RS. Functional venous admixture in the lungs of the turtle, Chrysemys scripta. Respir Physiol 1983;53:99 – 107. [27] Stinner JN. Ventilation, gas exchange and blood gases in the snake, Pituophis melanoleucus. Respir Physiol 1982;47:279–98. [28] Stinner JN. Thermal dependence of air convection requirement and blood gases in the snake Coluber constrictor. Am Zool 1987;27:41 – 7. [29] Stinner JN, Wardle RL. Effect of temperature upon carbon dioxide stores in the snake Coluber constrictor and the turtle Chrysemys scripta. J Exp Biol 1988;137:529 – 48. [30] Stinner JN, Hartzler LK, Grguric MR, Newlon DL. A protein titration hypothesis for the temperature-dependence of tissue CO2 content in reptiles and amphibians. J Exp Biol 1998;201:415 – 24. [31] Tucker VA. Oxygen transport by the circulatory system of the green iguana (Iguana iguana) at different body temperatures. J Exp Biol 1966;44:77 – 93. [32] Tucker VA. Method for oxygen content and dissociation curves on microliter blood samples. J Appl Physiol 1967;23:410–4. [33] Ultsch GR, Jackson DC. pH and temperature in ectothermic vertebrates. Bull Alabama Mus Nat Hist 1996;18:1 – 41. [34] Wang T, Abe AS, Glass ML. Non-linear Hill plots and low temperature sensitivity of whole blood from the rattlesnake Crotalus durissus and its implication for O2 transport in vivo. FASEB J. 1994;8:4858. [35] Wang T, Hicks JW. The interaction of pulmonary ventilation and the right-left shunt on arterial oxygen levels. J Exp Biol 1996;199:2121– 9. [36] Wang T, Smits A, Burggren WW. Lung function. In: Gans C, Gaunt AS, editors. Biology of Reptilia. 1998; in press. [37] Wood SC. Effect of O2 affinity on arterial PO2 in animals with central vascular shunts. J Appl Physiol 1982;53:1360 – 4. [38] Wood SC. Cardiovascular shunts and oxygen transport in lower vertebrates. Am J Physiol 1984;247:R3 – R14. [39] Wood SC, Hicks JW. Oxygen homeostasis in vertebrates with cardiovascular shunts. In: Johansen K, Burggren WW, editors. Cardiovascular Shunts. Phylogenetic, Ontogenetic and Clinical Aspects. Copenhagen: Munksgaard, 1985:354 – 72. [40] Zijlstra WG, Buursma A, Zwart A. Molar absorptivities of human hemoglobin in the visible spectral range. J Appl Physiol 1983;54:1287 – 91.
Effects of temperature on lung and blood gases in the South American rattlesnake Crotalus durissus terrificus Tobias Wang a,*, Augusto S. Abe a, Mogens L. Glass b b
a Department of Zoology, UNESP-Rio Claro, 13500 Rio Claro SP, Brazil Department of Physiology, School of Medicine, Uni6ersity of Sao Paulo, 14049 Riberao Preto SP, Brazil
Received 25 February 1998; received in revised form 3 May 1998; accepted 1 June 1998
Abstract The effects of temperature on lung and blood gases were measured in the South American rattlesnake (Crotalus durissus terrificus). Arterial blood and lung gas samples were obtained from chronically cannulated animals at 15, 25, and 35°C. As expected for reptiles, arterial pH fell with increased temperature (0.018 U °C − 1 between 15 and 25°C and 0.011 U °C − 1 between 25 and 35°C) while lung gas PCO2 rose from 5.8 mmHg at 15°C to 13.2 mmHg at 35°C. Concurrently, lung gas PO2 declined from 132 mmHg at 15°C to 120 mmHg at 35°C, and arterial PO2 increased from 33 to 76 mmHg in that temperature range. Arterial haemoglobin O2 saturation rose from 0.53 at 15°C to 0.83 at 25°C but became slightly reduced (0.77) with a further elevation of temperature to 35°C. Arterial haemoglobin concentration increased from 1.96 to 2.53 mM between 15 and 35°C, consistent with higher demands on oxygen delivery to tissues at elevated temperatures. Moreover, the substantial increase of haemoglobin O2 saturation between 15 and 25°C conforms to the idea that reduction of the central vascular right-to-left shunt (pulmonary bypass of systemic venous return) is associated with high metabolic demands. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Reptile; Snake; Crotalus; Temperature; Acid – base balance; O2 transport; Lung gases; Arterial blood gases
1. Introduction Many ectothermic vertebrates undergo large diurnal and seasonal fluctuations in body temperature with associated changes in metabolism and in cardio-respiratory function. The effects of body temperature on arterial blood gases have been described for a number of reptiles and amphibians and, as a general rule, increased temperature is associated with a reduction in arterial pH, an elevation of arterial PCO2, and a rise of arterial PO2 [7,30,33,39]. Wood [37,38] attributed the increase in arterial PO2 to a reduction of blood oxygen affinity with increased temperature in combination with an incomplete saturation of arterial blood. In most * Corresponding author. Present address: Center for Respiratory Adaptation, Institute of Biology, Odense University, 5230 Odense M, Denmark. Tel.: + 45 65 572460; fax: +45 65 930457; e-mail: [email protected]
amphibians and reptiles, the systemic and pulmonary circulations are not completely separated and the arterial blood is, therefore, a mixture of venous systemic blood and blood returning from the lungs (central right-to-left shunt; R–L shunt). As a consequence, the oxygen saturation of the arterial blood becomes reduced and arterial PO2 can be considered a dependent variable determined by the resulting haemoglobin O2 saturation and the blood oxygen affinity. Assuming that haemoglobin oxygen saturation (HbO2 saturation) is unaffected by temperature, PO2 increases with temperature due to right-shift of the oxygen dissociation curve [38]. Few studies report on the arterial blood oxygen saturation in relation to temperature in reptiles, but in the lizard Iguana iguana and the snake Pituophis melanoleucus, HbO2 saturation is constant and temperature-independent over a wide temperature range [27,31]. However, in turtles and varanid lizards, micro-
1095-6433/98/$ - see front matter © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)10102-2
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T. Wang et al. / Comparati6e Biochemistry and Physiology, Part A 121 (1998) 7–11
sphere injections document that the R – L shunt is reduced with increased temperature [9,15]. A reduction in the R–L shunt is expected to increase arterial HbO2 saturation and would contribute to the increased PO2 with elevated temperature. In the present study we provide direct measurements of arterial HbO2 saturation, haemoglobin concentration, PO2, pH, as well as lung gas pressures at three different temperatures in the South American rattlesnake Crotalus durissus terrifus.
2. Materials and methods
2.1. Experimental animals Six rattlesnakes (400 – 800 g) were obtained from the Butantan Institute (Sao Paulo, Brazil) and kept in outdoor enclosures at the campus of Universidade Estadual Paulista in Rio Claro (Sao Paulo, Brazil) until experimentation. The snakes were fed mice until 7–10 days before surgery.
2.2. Surgery Each snake was anaesthetised by inhalation of ether vapour until ciliary reflexes were absent and it ceased to breathe spontaneously. Then the trachea was intubated using a small rubber tube and the animals were ventilated manually every 2 – 3 min with a mixture of air and ether vapour to maintain anaesthesia. The coeliac artery was occlusively cannulated by advancing a PE50 catheter containing heparinized saline into the dorsal aorta. In addition, a pulmonary catheter (PE 160 with a flared end and sideholes) was placed with the tip protruding 0.2 – 0.5 cm into the lumen of the vascularized portion of the lung. Subsequently, lung parenchyma was tied around the catheter, which was then fed out dorsally and secured to the back of the snake with a few sutures. Both incisions were closed with intermittent sutures and the snake was allowed to recover from the anaesthesia for 18–36 h at 25°C.
2.3. Analysis of blood and lung gases Arterial PO2 and pH were measured using a Radiometer (Copenhagen, Denmark) Blood Micro System that maintained the electrodes at the experimental temperature. Both electrodes were calibrated immediately prior to measurements. For the PO2 measurements an experimentally determined liquid/gas correction factor as well as the effects of blood metabolism were taken into account. Total oxygen content of the arterial blood ([O2]a) was measured as described by Tucker [32] taking the correc-
tion by Bridges et al. [1] into account. Haemoglobin concentration ([Hb]) was measured spectrophotometrically at 540 nm following conversion to cyanomethhaemoglobin and applying a millimolar extinction coefficient of 11.0 [40]. Because some previous studies on reptiles have reported high concentrations of nonfunctional haemoglobin [21], we obtained an in vitro relationship between the oxygen carrying capacity of the haemoglobin (HbO2cap) and the haemoglobin concentration ([Hb]) for each snake. To this purpose, a 1.5 ml blood sample was equilibrated in a rotating tonometer (Eschweiler, Germany) with a gas mixture of 30% O2 and 2% CO2 (balance N2) for 30 min, after which total oxygen content and [Hb] were determined as described above. From the measurement of O2 content, HbO2cap was calculated by subtracting dissolved O2 (aO2 · PO2, where aO2 is the human O2 solubility coefficient [2]). Having established the correlation between HbO2cap and [Hb], the arterial oxygen haemoglobin saturation (arterial HbO2 sat) was calculated as arterial HbO2 sat= 100 · [(CaO2 − (aO2 · PaO2))/ HbO2cap)]%. Lung gas samples of 5 ml were drawn into gas tight syringes and analysed immediately for O2 and CO2 concentrations by means of a Beckman Medical Infrared CO2 Analyzer (LB-2) and an Applied Electrochemistry Oxygen Analyzer (S-3A/L) connected in series. The analysers were calibrated with high precision gas mixtures from Aga (Brazil).
2.4. Experimental protocol Experiments were postponed for a minimum of 18 h after surgery. The snakes were maintained in a climatic chamber (Favolet, Sao Paulo, Brazil) that provided both visual and acoustical isolation of the animals throughout the measurements. Measurements were conducted at three different temperatures (15, 25, and 35°C). The experiments always started at 25°C, while the second experimental temperature was chosen at random and no measurements were taken within a 12 h period following a change of temperature. For each snake at each temperature, at least 20 lung gas and three arterial blood samples were obtained and analysed. The measurements of blood gases were interspersed by approximately 2 h, while the lung gas measurements were taken at approximately 15 min intervals. For each snake, a mean value of blood and lung gases was calculated on the basis of all the measurements at the respective temperatures. For the analysis of pH, [Hb], [O2]a, and PO2, a blood sample of 600 ml was withdrawn, but since some of the blood could be re-injected after analysis, each analysis was only associated with a blood loss of approximately 250 ml.
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2.5. Statistics A one-way ANOVA for repeated measures was employed to assess significant effects of temperature on the reported parameters. Differences among means were subsequently assessed using a Student-NewmanKeul’s test. A fiducial limit for significance of P 5 0.05 was applied and all data in the text and figures are presented as mean9 1 S.D.
3. Results Arterial pH decreased significantly by 0.0189 0.007 U °C − 1 between 15 and 25°C and by 0.01190.003 U °C − 1 between 25 and 35°C (Fig. 1A). This reduction in pHa reflects a significant increase in intrapulmonary PCO2 from 5.8 9 1.7 mmHg at 15°C to 13.2 9 2.2 mmHg at 35°C (Fig. 1B). Intrapulmonary PCO2 increased relatively more within the low temperature range compared to the high range; D log(PCO2)/DT was 0.23 between 15 and 25°C and only 0.12 between 25 and 35°C. Concurrent with the rise in PCO2, increased temperature was associated with a reduction in intrapulmonary PO2 that declined from 131.99 3.3 mmHg at 15°C to 119.5 95.7 mmHg at 35°C (Fig. 1C). Conversely, arterial PO2 rose from 32.795.8 mmHg at
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15°C to 66.79 3.4 at 25°C and reached 75.794.4 mmHg at 35°C (Fig. 1C). Based on the alveolar gas equation [20], effective ventilation (V: eff) relative to pulmonary CO2 output (V: CO2) was calculated as V: eff/V: CO2 = RT/PLCO2, Where PLCO2 is the PCO2 in the gas exchange zone of the lung, R is the gas constant, and T is the absolute temperature (K). Based on the mean values for PLCO2 obtained on Crotalus in the present study, V: eff/V: CO2 is predicted to decrease from 138 at 15°C to 65 at 35°C (Fig. 1D). Arterial HbO2 saturation increased significantly from 0.539 0.09 at 15°C to 0.83 9 0.03 at 25°C, while a further increase in temperature to 35°C was associated with a non-significant reduction in HbO2 saturation to 0.7790.08 (Fig. 1E). Also, the haemoglobin concentration of the arterial blood increased significantly with rising temperature from 1.969 0.15 mM at 15°C to 2.5390.20 mM at 35°C (Fig. 1F). The total oxygen carrying capacity of the blood determined in vitro increased correspondingly. For all snakes, the determinations of oxygen carrying capacity of the haemoglobin (HbO2cap) and the haemoglobin concentration ([Hb]) were within 9 3% of each other, indicating that virtually all haemoglobin was functional, i.e. that methhaemoglobin levels were insignificant.
4. Discussion
4.1. Effects of temperature on arterial pH The negative DpH/DT in Crotalus is consistent with data for most ectothermic vertebrates [10,33] and with reports for other species of snakes [3,19,27,30]. Reeves [24] suggested that the change in pH parallels that of pK for the alpha imidazole system such that the degree of protein ionization is maintained constant (alphastat regulation). In many species, the DpH/DT is less than required by the hypothesis [7,10,30]. In Crotalus, the pH change between 15 and 25°C was compatible with alphastat regulation, whereas the change was smaller than predicted between 25 and 35°C.
4.2. Effects of temperature on blood and lung gases
Fig. 1. The effects of temperature on lung and blood gases in the South American rattlesnake (Crotalus durissus terrificus). A, Arterial pH; B, Intrapulmonary PCO2 (PLCO2); C, Arterial PO2 and intrapulmonary PO2 (PLO2); D, Effective ventilation (Veff) relative to pulmonary CO2 output (V: CO2); E, Arterial haemoglobin oxygen saturation (HbO2 saturation); F, Arterial haemoglobin concentration ([Hb]). Mean values 9 1 S.D. (N =6).
The values for PO2 and PCO2 in the lung gas of Crotalus are consistent with direct measurements of lung gases from the viper Vipera anthina palestinae [8] and end-expired gases in the python Morelia spilotes [6]. In these species the PLO2 ranged from 120 to 145 mmHg and PLCO2 from 5 to 20 mmHg. The data for Vipera and Morelia were confined to the narrow temperature range of 22–26°C and the effects of tempera-
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ture were, therefore, not evaluated. In Crotalus a marked decrease of PLO2 from 132 mmHg (15°C) to 120 mmHg (35°C) was accompanied by increases of PaO2, whereby the PLO2 −PaO2 difference was reduced from about 100 mmHg to about 45 mmHg. Also, the increase of PaO2 with temperature in Crotalus (Fig. 1) is consistent with previous reports on snakes and represents a general pattern among reptiles and amphibians [7,19,27,29]. Wood [37,38] explained this pattern as a consequence of the R – L shunt. In its presence, arterial HbO2 saturation is reduced due to the admixture of O2 poor systemic blood to the O2 rich blood returning from the lungs, and PaO2 becomes a dependent variable determined by the resulting haemoglobin oxygen saturation and the blood oxygen affinity. Thus, as increased temperature lowers blood oxygen affinity, PaO2 will increase even if haemoglobin oxygen saturation is constant. In Crotalus, however, the arterial HbO2 saturation increased markedly between 15 and 25°C and thus contributes to the pronounced elevation of PaO2 within this temperature range. In contrast to the large increase in HbO2 saturation observed in Crotalus, two previous studies on snakes and iguanas report a temperature-independent HbO2 saturation over a wide temperature range [27,31]. In reptiles, the PLO2 −PaO2 difference is the combined result of diffusive resistance of the barrier separating lung gas and blood, ventilation-perfusion inhomogeneity within the lung and intrapulmonary and central vascular R– L shunts [36]. Since a change in some or all of these factors will alter arterial HbO2 saturation, it is difficult to establish the mechanism behind the large increase of HbO2 saturation between 15 and 25°C in the present study. In sea snakes, an intrapulmonary shunt of almost 20% has been determined [25]. Similar values have been reported for turtles, tegu lizards, and alligators [12,14,22,26], while values for monitor lizards are substantially smaller [13]. In turtles, the pulmonary shunt flow increases whenever pulmonary blood flow increases at constant temperature [14], but no studies have addressed this aspect in relation to temperature. Because the ophidian lung consists of an anterior, highly vascularized section and a posterior, virtually non-vascularized region [18], ventilation-perfusion inhomogeneity and stratification also increase the PLO2 − PaO2 difference [5,6,8,23]. However, these factors probably contribute little to the PLO2 − PaO2 difference in resting animals [13,14]. Therefore, it seems reasonable to suggest that a reduction in cardiac R–L shunt with increased temperature in Crotalus can explain most of the increased HbO2 saturation between 15 and 25°C. Consistent with this view, a decrease of the cardiac R – L shunt with rising temperature has been reported for the turtle Chrysemys picta bellii [9] and the monitor lizard Varanus niloticus [15]. This finding is compatible with the hypothesis that
reptiles reduce the magnitude of R–L shunt during conditions of elevated metabolic rate, whether due to exercise, digestion, or increased temperature [11,35]. In addition to reduction in R–L shunt, the large increase in blood [Hb] and, consequently, in blood oxygen carrying capacity, of Crotalus promotes oxygen delivery at higher temperatures.
4.3. A possible conflict between acid–base balance and O2 transport at high temperatures The control of pulmonary ventilation performs the dual role of regulating arterial acid–base balance through adjustments of PCO2 and of regulating arterial oxygen levels. Potentially, a conflict between these functions may develop in amphibians and reptiles exposed to high body temperature. The present and most previous studies show that arterial pH decreases with increased body temperature [30]. This negative DpH/DT is achieved by ventilatory adjustments to elevate PaCO2, although changes in bicarbonate levels may contribute [29,33]. The increased PaCO2 is accomplished through a progressive decrease in effective ventilation relative to metabolic rate as temperature increases [3,4,16,19,27]. As a consequence of the progressive hypoventilation with increased temperature, intrapulmonary PO2 is progressively reduced (Fig. 1, [28]). This decline of PLO2 coincides with a reduction in blood oxygen affinity and must at some point reduce the HbO2 saturation of the blood leaving the lung. On this basis and using the two-compartment model, it has been predicted that there exists a critical temperature above which arterial oxygen levels (PO2 and HbO2 saturation) decrease due to impaired oxygen loading at the lungs [39]. Some experimental data support the existence of such a ‘breaking point.’ In the snake Coluber constrictor, arterial O2 content decreased upon exposure to body temperatures above 30°C [28]. Likewise, in the turtle Pseudemys floridana, both pulmonary venous and systemic arterial HbO2 saturation decreased with rising temperature [17]. The blood oxygen affinity of Crotalus is very low (P50 is approximately 50 mmHg at 30°C, pH 7.42 [34]). Accordingly, this species should be susceptible to this conflict. However, the high PLO2 (115 mmHg) at 35°C undoubtedly contributes to maintaining adequate arterial O2 levels. Moreover, a direct evaluation of impaired blood oxygen binding at high temperatures would require direct measurements of HbO2 saturation of the pulmonary venous blood. In conclusion, PaO2 increased with rising temperature, whereas lung gas PO2 decreased but still remained high. HbO2 saturation increased markedly between 15 and 25°C, suggesting a reduction in the cardiac R–L shunt. There was no unequivocal evidence for a conflict between oxygen transport and acid–base regulation in Crotalus exposed to high temperature. Clear signs of a
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conflict may require more strenuous conditions such as a high metabolic rate during exercise or digestion, or during hypoxia.
Acknowledgements This study was supported by an equipment grant from FAPESP (89/2857-5) and CNPq (300603/91-6). T. Wang received support from the Danish Research Council.
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