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© 2002 International Anesthesia Research Society
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Abstract |
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Because perfluorocarbon (PFC) liquid contacts closely with the alveolar capillaries during partial liquid ventilation (PLV), PLV with cold PFC may be used for the induction of hypothermia. Twenty rabbits were randomized to PFC-induced hypothermia (PH) (n = 7; core temperature 35° ± 1°C), surface hypothermia (SH) (n = 7; 35° ± 1°C), or normothermia (n = 6; 39° ± 1°C). We induced PH by repeated in situ exchanges of 0°C perfluorodecalin during PLV. At the establishment (0 min) of hypothermia in the PH group, oxygen consumption (P = 0.04) and oxygen extraction ratio (P = 0.01) decreased from normothermic condition. Metabolic (oxygen consumption, oxygen extraction ratio, serum lactate level) and hemodynamic variables (heart rate, blood pressure, cardiac output, pulmonary artery pressure) of the PH group were not different from those of the SH group at 0, 30, 60, 90, and 120 min of hypothermia. The difference in temperature between the pulmonary artery and rectum during the hypothermic period was smaller in the PH group compared with the SH group (P = 0.033). In conclusion, hypothermia may be induced during PLV by using cold PFC. This "pulmonary method" of cooling was comparable to a systemic method of cooling with regard to a few important physiologic variables, while maintaining a narrower interorgan temperature difference.
IMPLICATIONS: The induction of moderate hypothermia was feasible in rabbits by administrating cold perfluorocarbon liquid into the lung. Physiologic changes induced by this pulmonary cooling were comparable to those induced by systemic cooling. Our method may be regarded as a methodological advance in the field of therapeutic hypothermia.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induced hypothermia is used in various fields of medicine, e.g., cardiac surgery, ex vivo organ/tissue preservation, brain injury, sepsis/acute respiratory distress syndrome, with the purpose of preserving tissue integrity in the setting of compromised blood supply or exaggerated inflammation (1–5). Among various methods of induced hypothermia, body surface cooling is the one most commonly used at the bedside. In this method, part of the systemic circulation (cutaneous blood) is first cooled and the core body is cooled later in a retrograde manner.
Contrary to this systemic method, hypothermia may also be induced in an anterograde way by first cooling the pulmonary circulation (6–9), to be called a pulmonary method for induced hypothermia. The lung can serve as a core heat exchanger because (1) the entire cardiac output (CO) passes through the pulmonary capillary network and (2) the heat of the body can be transferred to the environment via the vast alveolar surface. Despite these theoretical merits, a practical method of pulmonary cooling has not been available at the bedside.
Partial liquid ventilation (PLV), an investigational ventilatory method for severe respiratory failure (10,11), permits a perfluorocarbon (PFC) liquid to contact closely with the alveolar capillaries (12). Because PFC liquid carries a high thermal conductivity (e.g., 0.1 kcal/h · m · °C at 25°C with PFC RIMAR 101 [Miteni, Milan, Italy]), it is conceivable that an administration of cold PFC liquid to the lung may be used to induce hypothermia. Although total liquid ventilation with PFC has been studied for this purpose (8,9), such a trial has not been reported for PLV, which seems closer to clinical application than the former method that requires an extracorporeal circuit. We designed this study to determine the feasibility of cold PFC-induced hypothermia (PH) during PLV and to compare its physiologic characteristics with those of a systemic method of cooling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal Preparation and Surgical Procedures
Twenty-two healthy New Zealand White rabbits (3.1 ± 0.2 kg) were used for this study. The rabbits were cared for and handled according to the guidelines of the US National Institutes of Health. Any rabbit that was considered ill by our institution’s veterinarian was precluded from this study. In addition, rabbits were also excluded from the study if they showed metabolic acidosis (pH < 7.35 with base excess less than -5 mEq/L), or leukocytosis (white blood cell [WBC] count >7000/mm3) (13) at the completion of the surgical procedures under general anesthesia (n = 2). The eligible 20 rabbits were randomized to PH (n = 7), surface hypothermia (SH) (n = 7), or normothermia (n = 6). The ambient temperature of the animal laboratory room was maintained at 22° ± 1°C. The surgical preparation and experimental protocol were approved by our institution’s Animal Care Committee.
After an IM injection of a mixture of xylazine (5 mg/kg) and ketamine (25 mg/kg) into the thigh, a peripheral ear vein was secured with a 24-gauge angiocath for an IV route. A tracheostomy was performed after a midline incision of the neck, and a 4-mm (internal diameter) cuffless endotracheal tube was inserted a distance of 4 cm into the trachea and tied firmly to prevent gas/liquid leak. The carotid artery was cutdown and cannulated with a 22-gauge angiocath for arterial blood sampling and for monitoring arterial pressure and pulse rate with a pressure monitor, Escort II (Medical Data Electronics, Arleta, CA). A 4F pulmonary artery (PA) catheter (Arrow International, Reading, PA) was inserted through the right internal jugular vein to the PA for venous blood sampling, determination of PA pressure, and measurement of CO. The PA catheter was advanced up to the PA under the guidance of pressure profile. The injectate opening of the catheter (10 cm from the tip of the catheter) was confirmed to be within the right atrium by observation of the corresponding pressure profile.
Anesthesia was maintained by an IV infusion of a mixture of ketamine (75 mg) and xylazine (5 mg) in 20 mL of normal saline at a rate of 0.4–0.6 mL/min through the ear vein. Muscle paralysis was maintained during the study with intermittent IV administrations of pancuronium (0.1 mg/kg). Mechanical ventilation was performed by using a Servo 300 ventilator (Siemens-Elema, Solna, Sweden) with the following variable values: tidal volume (VT) 10 mL/kg, frequency 35/min (adjusted to obtain a PaCO2 of 35–45 mm Hg at the baseline), fractional concentration of inspired oxygen (FIO2) 1.0, positive end-expiratory pressure 3 cm H2O, inspiratory-to-expiratory ratio 1:1 by setting inspiratory time at 30% and inspiratory pause at 20% of a respiratory cycle. The ventilator variables were kept unchanged throughout the experiment. An IV fluid (5% dextrose in 0.45% saline) was given by an infusion pump at a rate to keep the vein open (7.5 mL · kg-1 · h-1).
Induction and Maintenance of Normothermia and Hypothermia
The target core temperatures (Tcore) were 39° ± 1°C for normothermia and 35° ± 1°C for hypothermia, a 4°C difference, which is comparable to the strategy of mild hypothermia in humans (4,5). Body temperature was determined simultaneously from the PA (Tcore) by a thermistor at the distal end of the PA catheter, and from 5-cm deep in the rectum by a rectal probe (intermediate or shell temperature, Tshell) (14).
PH was induced by administering 0°C perfluorodecalin (perfluor-decahydronaphthalin, C10F18) (Fluka Chemie AG, Buchs, Switzerland) into the lung. The first dose of PFC, 30 mL/kg, was divided into two equal doses, which were administered to each lung at the corresponding lateral decubitus position. As soon as the PA temperature stopped decreasing, the liquid was removed from the lung by a 1-m-long siphon, and replaced by new 0°C PFC at 20 mL/kg aliquots. This in situ PFC exchange was repeated until the target temperature was reached (10 to 12 exchanges in total; time to 35°C: 38 ± 4 min). Tcore of the PH group remained stable once the target temperature was established, and no further exchange of the liquid was necessary. The evaporative loss of PFC was not replaced during the 2-h period of hypothermia.
SH was induced by putting ice slush on the body surface of the rabbit except over the chest and abdomen (time to 35°C: 23 ± 3 min). As the Tcore approached 36°C, ice slush was removed from the animal, lest the Tcore decrease too far below the lower limit (34°C). During the 2-h period of hypothermia, ice slush was intermittently applied on the sides of the animal’s trunk because the temperature tended to increase toward the upper limit of temperature (36°C). In the Normothermia group, the Tcore was maintained by an electrical heating blanket.
Physiologic Measurements
When the rabbit was stable after surgical procedures and instrumentation, mean blood pressure (BP), mean PA pressure, heart rate (HR), CO, hemoglobin (Hb), WBC count, serum lactate, and blood gases were determined as indices of normothermic condition. As soon as the target temperature was attained, the same physiologic data were redetermined (0 min of hypothermia), and then every 30 min for 2 h. Blood gases were analyzed simultaneously with 0.3-mL specimens of arterial and mixed venous blood using standard electrodes, Blood Gas System 288 (Ciba-Corning, Medfield, MA) corrected for the actual Tcore. Hb and WBC count were determined with 1 mL of arterial blood by using a blood cell counter, SE-9000 (Sysmax Co., Kobe, Japan). Blood specimens for serum lactate were chilled immediately in an ice bucket, and transported within 5 min to our institution’s chemistry laboratory. Serum lactate was determined with 1.5 mL of arterial blood according to the lactate oxidase method by using the Vitros 750XRC Chemistry System (Johnson and Johnson Clinical Diagnostics Inc., Rochester, NY; reference range 7–19 mg/dL as determined at 540 nm). CO was the mean of 3 thermodilution measurements determined with 1.5 mL of normal saline (1° ± 1°C) using a CO computer, COM-1 (Baxter Healthcare Corp., Irvine, CA) with computation constant 0.054. Derived indices of oxygen metabolism were calculated as follows:
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where CavO2 is oxygen content, SavO2 is oxygen saturation, and PavO2 is partial pressure of oxygen of the arterial or mixed venous blood, respectively.
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Homogeneity of internal heat distribution during the hypothermic period was assessed with the absolute difference in temperature between the PA and rectum (
[Tcore - Tshell]) of all 5 time points (0, 30, 60, 90, and 120 min).
All data were expressed as mean ± SD unless otherwise stated. Comparison between unpaired values was performed by using the Mann-Whitney U-test. Statistical significance of the values between different time points within a group was evaluated by using Friedman analysis. Post hoc multiple comparisons were performed by using the Wilcoxon’s signed rank sum test using Bonferroni correction. A P value < 0.05 was considered statistically significant.
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Results |
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Physiologic Changes Induced by PH
At 0 min of hypothermia in the PH group, both
O2 (P = 0.04) and O2 ER (P = 0.01) decreased from normothermic condition, whereas serum lactate levels did not change significantly (Table 1). Mean PA pressure increased from normothermic condition (P = 0.01), whereas other hemodynamic variables did not change significantly (Table 2). Hb (10.1 ± 1.5 g/dL at normothermic condition, 10.3 ± 1.2 g/dL at 0 min of hypothermia; P = 0.233) and WBC count (3.7 x 103/mm3, 3.6 x 103/mm3, respectively; P = 1.0) were not changed by PH.
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Comparison of the Physiology Between the Two Methods of Hypothermia
Compared with the Normothermia group,
O2 (Fig. 1) and O2 ER (Fig. 2) were lower in both hypothermia groups at all time points (all P < 0.05).
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O2, O2 ER, and serum lactate levels (Table 1) of the PH group were not different from those of the SH group at all time points of hypothermia. Arterial pH (all P < 0.05) and PaO2 (all P < 0.05) were lower, and PaCO2 was higher (all P = 0.008) in the PH group compared with the SH group, at all time points of hypothermia. Mean BP, mean PA pressure, HR, and CO of the PH group were not different from those of the SH group at all time points of hypothermia (Table 2). Mean PA pressure decreased over time in the PH group (P = 0.003), whereas it did not change in the SH group.
Homogeneity of Internal Heat Distribution Between the Two Methods of Hypothermia
Tcore of all time points of hypothermia was 34.5° ± 0.5°C in the PH group and 34.4° ± 0.7°C in the SH group (P = 0.499). [Tcore - Tshell] during the hypothermic period was smaller in the PH group (0.3° ± 0.3°C) compared with the SH group (0.7° ± 0.9°C) (P = 0.033).
Complications
One rabbit each in both hypothermia groups developed pulmonary hypertension during the induction of hypothermia, and died. These animals were excluded from data analysis. Once hypothermia was established, no further episodes of pulmonary hypertension or ventricular fibrillation were noted in the remainder of the animals of both hypothermic groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study with healthy rabbits, PH in conjunction with PLV resulted in a reduced tissue metabolism without significant hemodynamic deterioration. With this pulmonary method of core cooling, both the metabolic characteristics (
O2, O2 ER, serum lactate level) and the hemodynamic physiology (HR, CO, BP, and PA pressure) were comparable to those of a systemic method (SH). The [pulmonary arterial-rectal] difference in temperature was smaller in the PH group compared with the SH group. In the past, the induction of therapeutic hypothermia based on cooling of the pulmonary circulation was only a theoretical possibility. Insufflation of cold gas to this purpose was found to be impractical (6,7) because a convective heat loss from the distal airways is limited by bronchoconstriction brought on by cold gas itself (15). Intrapulmonary liquid, however, was a feasible pulmonary method for an induction of hypothermia in small animals by Shaffer et al. (8) and Forman et al. (9). In their method with newborn lambs, total liquid ventilation was used to induce hypothermia, in which PFC liquids of 20°–30°C were tidally exchanged from the lung by an extracorporeal circuit (9). Our study is the first to demonstrate an induction of hypothermia by adopting PLV (no need of an extracorporeal circuit) and 0°C PFC. Interestingly, hypothermia established by this technique remained within the target temperature for the study period without the need of further exchange of the liquid. This aspect contrasted with surface cooling that necessitated intermittent application of ice over the body of the animal to maintain the desired temperature. Regarding the mechanism, we speculate that cold PFC liquid that resided in the core body (lung) could have served as "cold reservoir," which surface cooling lacked.
The effects of PH on some important metabolic variables (O2, O2 ER, and serum lactate level) were almost identical in direction and magnitude with the corresponding values obtained by SH. In particular, the decreased O2 ER at a similar CO to normothermic condition indicated that the metabolic rate of peripheral tissue was also effectively reduced by PH.
In our study, however, a few differences were noted between PH and SH. First, the difference between the core and intermediate or "shell" temperatures (14) during the hypothermic period was smaller with PH than with SH. In the latter method, or a systemic method of hypothermia, cooling of the body core is accom-plished in a retrograde way as a consequence of cooled cutaneous blood (accounting for approximately 10% of the CO in humans). In contrast, during a pulmonary method of hypothermia, the body core is first cooled and cooling would propagate in an anterograde direction, in which all organs/tissues would be exposed simultaneously to hypothermic blood amounting to the whole CO. In this situation, any uneven cooling of the body would be minimized. Body temperature is known regionally to be heterogeneous and becomes more so during cooling (14), and an uneven cooling incurred during the induction of hypothermia is known to cause metabolic acidosis (16). Our results therefore suggest that PH may allow a narrower interorgan temperature difference during hypothermia than a systemic method of cooling.
Second, some blood gas values (notably arterial pH, PaCO2, PaO2) were altered in PH, but not in SH. In the normal lung, as in our experiment, oxygenation during PLV is inferior to gas ventilation (12,17). Furthermore, because PFC is a diffusion barrier for CO2 (17), PaCO2 usually increases during PLV (18). Lower pH with hypercapnia during PH as opposed to SH was therefore attributable to PLV per se, rather than to the PH. Indeed, considering the decreased O2 during PH, it would be interesting to see whether hypothermic PLV can attenuate the complication of hypercapnia of PLV that is performed at normal body temperature.
During the PH in our study, systemic hemodynamic variables (HR, CO, and BP) were stable when compared with the normothermic condition. Despite wide differences in target temperature and species, previous studies have shown that during mild-to-moderate hypothermia, changes in BP depend on the balance of the changes in stroke volume, peripheral vascular resistance, and HR (19–21). In the mild hypothermia of our study, BP was preserved, because these determinants were not changed.
In the PH group, PA pressure was increased with the induction of hypothermia. Pulmonary vascular resistance could have been increased by pulmonary venous constriction and/or increased blood viscosity associated with hypothermia itself, or because of acidosis (19,22,23). Additionally, PFC itself might have contributed to the increase in PA pressure (24). This possibility was inferred from the gradual decrease of PA pressure with time, probably reflecting an evaporative loss of PFC liquid from the lung. Nevertheless, the initial increase of PA pressure in the PH group cautions against instituting this method of cooling in the presence of pulmonary hypertension. Fatal ventricular fibrillation, another common cause of cardiac morbidity/mortality after hypothermia (both induced and accidental), was not observed in the present study.
In our study, the results of arterial blood gas analysis were corrected for temperature. Although a temperature-uncorrected blood gas analysis is considered convenient for managing a hypothermic subject based on the 
-stat protocol (25,26), temperature-corrected values are preferred for the calculation of oxygen delivery and the evaluation of cardiopulmonary function (27). PH was not without complication in the present study. Although the majority of animals showed only a mild increase of PA pressure, one rabbit developed severe pulmonary hypertension, which was probably responsible for the fatality. Because of the difficulty of proper ballooning of the PA catheter we used, PA occlusion pressure could not be recorded in all of the rabbits. A more reliable method of occlusion is needed for the calculation of pulmonary vascular resistance in this small animal. Our present study on PH was limited with regard to the degree of hypothermia (mild degree), species of animal (small mammal), and to endured time of hypothermia (two hours). The feasibility and physiology of a deeper and longer hypothermia during PLV is open to future investigation in different species, including humans.
In conclusion, hypothermia may be induced during PLV by cooling the pulmonary circulation with cold PFC. This pulmonary method of cooling was comparable to a systemic cutaneous method of cooling with regard to O2, O2 ER, serum lactate level, and major hemodynamic variables, while maintaining a more homogeneous distribution of internal heat. If applied to a normal lung, however, the decreased efficiency of gas exchange of PLV as compared with gas ventilation may be a disadvantage of PH.
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Acknowledgments |
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This study was supported by Fund 1999-0367 from the Korea Research Foundation, Seoul, Korea.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- Kirklin JW, Kirklin JK, Lell WA. Cardiopulmonary bypass for cardiac surgery. In: Sabiston DC Jr, Spencer FC, eds. Gibbon’s surgery of the chest. 4th ed. Philadelphia: Saunders, 1983: 909–25.
- Garzon AA, Cheng C, Pangan J, et al. Hypothermic hyperbaric lung preservation for twenty-four hours with replantation. J Thorac Cardiovasc Surg 1968; 55: 546–54.[Web of Science][Medline]
-
Marion DW, Penrod LE, Kelsey SF, et al. Treatment of traumatic brain injury with moderate hypothermia. N Engl J Med 1997; 336: 540–6.
[Abstract/Free Full Text] - Blair E, Buxton RW, Cowley RA, et al. The use of hypothermia in septic shock. JAMA 1961; 178: 916–9.[Web of Science][Medline]
- Villar J, Slutsky AS. Effects of induced hypothermia in patients with septic adult respiratory distress syndrome. Resuscitation 1993; 26: 183–92.[Web of Science][Medline]
-
Zikria BA, Ferrer JM, Malm JR. Pulmonary hypothermia in dogs. J Appl Physiol 1968; 24: 707–10.
[Free Full Text] - Harrison MR, Hysing ES, Bo G. Control of body temperature: use of respiratory tract as a heat exchanger. J Pediatr Surg 1977; 12: 821–8.[Web of Science][Medline]
- Shaffer TH, Forman DL, Wolfson MR. Physiological effects of ventilation with liquid fluorocarbon at controlled temperatures. Undersea Biomed Res 1984; 11: 287–98.[Web of Science][Medline]
- Forman DL, Bhutani VK, Tran N, et al. A new approach to induced hypothermia. J Surg Res 1986; 40: 36–42.[Web of Science][Medline]
- Fuhrman BP, Paczan PR, DeFrancisis M. Perfluorocarbon-associated gas exchange. Crit Care Med 1991; 19: 712–22.[Web of Science][Medline]
-
Hirschl RB, Pranikoff T, Wise C, et al. Initial experience with partial liquid ventilation in adult patients with the acute respiratory distress syndrome. JAMA 1996; 275: 383–9.
[Abstract/Free Full Text] -
Leach CL, Greenspan JS, Rubenstein SD, et al. Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome. The LiquiVent Study Group. N Engl J Med 1996; 335: 761–7.
[Abstract/Free Full Text] - Mitruka BM, Rawnsley HM. Clinical biochemical and hematologic reference values in normal experimental animals and normal humans. 2nd ed. New York: Masson Publishing USA, 1981.
- Kurz A, Sessler DI, Christensen R, et al. Heat balance and distribution during the core-temperature plateau in anesthetized humans. Anesthesiology 1995; 83: 491–9.[Web of Science][Medline]
- Webb P. Cold exposure. In: Bennett PB, Elliot DM, eds. The physiology and medicine of diving. London: Bellaire Tindall, 1975.
- Brewin EG. Physiology of hypothermia. Int Anesthesiol Clin 1964; 58: 803–27.
-
Mates EA, Hildebrandt J, Jackson JC, et al. Shunt and ventilation-perfusion distribution during partial liquid ventilation in healthy piglets. J Appl Physiol 1997; 82: 933–42.
[Abstract/Free Full Text] -
Wolfson MR, Kechner NE, Roache RF, et al. Perfluorocarbon rescue after surfactant treatment: effect of perfluorocarbon dose and ventilatory frequency. J Appl Physiol 1998; 84: 624–40.
[Abstract/Free Full Text] - Black PR, Devanter SV, Cohn LH. Effects of hypothermia on systemic and organ system metabolism and function. J Surg Res 1976; 20: 49–63.[Web of Science][Medline]
- Zarins CK, Skinner DB. Circulation in profound hypothermia. J Surg Res 1973; 14: 97–104.[Web of Science][Medline]
- Frank SM, Higgins MS, Breslow MJ, et al. The catecholamine, cortisol, and hemodynamic responses to mild perioperative hypothermia. Anesthesiology 1995; 82: 83–93.[Web of Science][Medline]
- Pennington DG, Hyman AL, Woolverton WC. Pulmonary vascular responses to selective lung cooling in intact dogs. Proc Soc Exp Biol Med 1971; 137: 1375–80.[Medline]
- Bergofsky EH. Mechanisms underlying vasomotor regulation of regional pulmonary blood flow in normal and disease states. Am J Med 1974; 57: 378–94.[Web of Science][Medline]
-
Lowe CA, Shaffer TH. Pulmonary vascular resistance in the fluorocarbon-filled lung. J Appl Physiol 1986; 60: 154–9.
[Abstract/Free Full Text] -
Swain JA. Hypothermia and blood pH. Arch Intern Med 1988; 148: 1643–6.
[Abstract/Free Full Text] - McConnell DH, White FN, Nelson RL, et al. Importance of alkalosis in maintenance of "ideal" blood pH during hypothermia. Surg Forum 1975; 26: 263–5.[Web of Science][Medline]
- Fromm RE Jr Guimond J-G, Darby J. The craft of cardiopulmonary profile analysis. In: Snyder JV, Pinsky MR, eds. Oxygen transport in the critically ill. Chicago: Year Book Medical Publishers, 1987: 249–69.
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