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Departments of *Anesthesiology and Intensive Care Medicine and Pathobiochemistry, Institute for Pathophysiology and Pathobiochemistry, Department of Neurosurgery and Institute for Pharmacology and Toxicology, Friedrich-Schiller-University, Jena; and || Institute of Pharmacology, Ernst Moritz Arndt University, Greifswald, Germany
Address correspondence and reprint requests to Harald G. Fritz, MD, Department of Anesthesiology and Intensive Care Medicine, Martha-Maria Hospital Halle D*lau gGmbH, Roentgenstr. 1, D-06120 Halle/S., Germany. Address e-mail to harald_fritz_2000{at}yahoo.de.
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Abstract |
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Introduction |
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Background anesthesia seems to be an independent factor that may influence both the putative protective and detrimental effects of hypothermia. Indeed, hypothermia itself may alter the pharmacokinetics of anesthetics. The plasma concentration of propofol increases about 28% during mild hypothermia in humans (4). During reduced core temperature, a doubling of vecuronium-induced neuromuscular block duration has been demonstrated in humans (5). Additionally, the anesthetic/analgesic regimen after TBI may dramatically influence the response to hypothermia (6).
The synthetic opioid fentanyl, when administered IV, is cleared predominantly by hepatic biotransformation (7). Fentanyl is metabolized by the microsomal cytochrome P450 3A4 (CYP3A4) isoform in humans (8), which is also present in the pig liver with similar activity (9). However, the effects of mild hypothermia on the action of continuously administered opioids for long-term analgesia and sedation (e.g., fentanyl) are not well understood.
We therefore examined, in a porcine model, the effect of mild hypothermia (32°C) on the plasma concentration of fentanyl during continuous long-term administration (33 h). We also investigated the effect of hypothermia on perfusion of organs that are involved in fentanyl metabolism and excretion, using color-labeled microspheres (CMS) (10). Furthermore, we determined temperature dependency of porcine hepatic microsomal CYP3A4 activity in vitro by ethylmorphine N-demethylation. We studied whether a temporarily decreased and subsequently reestablished body temperature, by altering the enzyme activity responsible for hepatic biotransformation, would influence the plasma concentration of fentanyl during continuous infusion.
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Methods |
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In Vivo Study
Seven female juvenile pigs of mixed German domestic breed (6-wk-old, weight: 11.8 ± 1.1 kg) were initially sedated with ketamine hydrochloride (20 mg/kg) and midazolam (1 mg/kg), and anesthesia was maintained during surgery by 70% nitrous oxide in 30% oxygen and 2% isoflurane. After tracheotomy (tube size: 5.5-mm inner diameter) and insertion of a central venous catheter into the left external jugular vein for drug administration and fluid therapy (lactated Ringer's solution: 4 mL · kg–1 · h–1), muscular relaxation was achieved with IV pancuronium bromide (0.4 mg · kg–1 · h–1). The lungs of the pigs were mechanically ventilated using pressure-controlled ventilation (Servo Ventilator 900C; Siemens-Elema, Solna, Sweden) with an oxygen/air mixture (Fio2 0.35). Ventilation was controlled by continuous end-expiratory CO2 monitoring and arterial blood gas check with 
-stat regime hourly.
The left brachial artery was cannulated for continuous monitoring of mean arterial blood pressure (MABP). A thermodilution catheter for cardiac index (CI) measurement was inserted into the abdominal aorta 2 cm below the diaphragm via the femoral artery. A left atrium catheter (inner diameter 1.0 mm) was placed via a left thoracotomy for CMS injection. In addition, a catheter (inner diameter 0.5 mm) was inserted into a branch of the pulmonary artery for mixed venous blood sampling. Another catheter (inner diameter 1.4 mm) was advanced from the left femoral artery into the abdominal aorta in order to withdrawal reference samples during regional blood flow measurement (see below).
An electrocardiogram was recorded from standard limb leads using stainless steel needle electrodes. Body temperature was controlled by a rectal thermoprobe, maintained throughout instrumentation at 37.5° ± 0.5°C using a water-filled pad connected to a heating-cooling thermostat and a feedback-controlled heating lamp. Physiological variables were recorded on a multichannel polygraph (MT95K2; Astro-Med).
Experimental Protocol and Management of Hypothermia.
After instrumentation, anesthesia was maintained throughout the experiment with a continuous infusion of fentanyl (15 µg · kg–1 · h–1) and midazolam (1.0 mg · kg–1 · h–1). During the baseline stabilization period of 12 h, values were recorded at 6 h (measuring point one, MP1) and 12 h (MP2). Systemic hypothermia was started 1 h after MP2 by means of a cooling blanket and forced air cooling. Hypothermia was guided by a rectal thermocouple probe. The animals were surface cooled within 217 ± 101 min to a core temperature of 31.6° ± 0.2°C for 6 h (MP3). Afterward, animals were rewarmed (MP4) within 258 ± 55 min and kept normothermic at 37.7° ± 0.3°C for a further 6 h (MP5) (Fig. 1).
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Measurements.
Blood samples for analysis of plasma fentanyl concentrations were injected into glass tubes. The plasma was separated in a refrigerated centrifuge (4°C) and placed in polypropylene tubes. Plasma fentanyl concentrations were measured by radioimmunoassay (Fa. Janssen Biotech, Olen, Belgium) at the five time points (MP1–5) (11). The assay sensitivity was 0.05 ng/mL, with intra- and intercoefficients of variation 6.0% and 6.9%, respectively. The assay was specific for fentanyl and did not include the metabolites.
Organ blood flows were measured using the reference sample CMS technique (10). Briefly, a known amount (approximately 3 x 106 per injection) of polystyrene CMS (diameter: 15.5 ± 0.33 µm) in 0.01% Tween 80, surface coated with 1 of 5 dyes (blue, yellow, white, red, violet) (Dye-Trak; Triton Technology, San Diego, CA) were thoroughly vortexed and sonicated and immediately injected within 20 s into the left atrium. CMS injected had a different color for each measuring point (MP1–5). A blood sample was withdrawn from the descending aorta as the reference sample, beginning 15 s before the CMS injection and continuing for 2 min at a rate of 3 mL/min (syringe pump SP210iw; World Precision Instruments, Inc., Sarasota, FL). The CMS injection did not alter MABP. At the end of each experiment, the pigs were killed with potassium chloride and the organ tissue samples from pancreas, spleen, liver, kidney, stomach (cardia, fundus, and pylorus, 4–6 g each), and gut (duodenum, jejunum, and ileum, 4–6 g each) were removed for processing. Blood and tissue samples were digested and the dye content of the contained CMS was estimated by photometric absorption with a diode-array ultraviolet/visible spectrophotometer (model 7500, wave length range 300–800 nm with a 2-nm optical band width; Beckman Instruments, Fullerton, CA). Calculations were performed using the MISS software (Triton Technology). The number of CMS was calculated using the specific absorbency value of the different dyes (provided by the manufacturer). Absolute tissue blood flows measured by CMS were calculated by the formula: flowtissue = number of microspherestissue · (flowreference/number of microspheresreference). Flows are expressed in mL/min per 100 g of tissue.
Assuming the oxygen capacity of hemoglobin to be 1.39 mL O2/g hemoglobin in pigs, blood O2 content was calculated as equal to grams of hemoglobin/mL · 1.39 mL O2/g hemoglobin · O2 saturation and expressed in mL/100 mL. Dissolved oxygen was added by calculation, using the measured Po2 and the temperature-corrected solubility coefficient for oxygen. The systemic oxygen consumption (
o2) was calculated by multiplying CI by the difference in arteriovenous O2 content. Systemic oxygen extraction rate was calculated by arteriovenous O2 content divided by arterial oxygen content.
In Vitro Study
Biological Material.
To determine temperature dependency of CYP3A4 activity, 9000g supernatants from pig liver specimens were used. Four juvenile pigs of mixed German domestic breed (age: 6 wk, weight: 12.7 ± 1.5 kg) were initially sedated with ketamine hydrochloride (20 mg/kg) and midazolam (1 mg/kg) and afterward killed for organ removal by IV injection of 10 mL of saturated magnesium chloride. Subsequently, the liver specimens (approximately 1 g each) were quickly removed and shock-frozen in liquid nitrogen and kept therein until processing. For preparation of the 9000g supernatants, the specimens were homogenized in ice-cold 0.1 M sodium phosphate buffer pH 7.4 (1:3 w/v) and centrifuged at 9000g for 20 min at 4°C. The protein content of the supernatants was determined by modified Biuret method.
Cytochrome P450-Dependent Monooxygenase Model Reactions.
Ethylmorphine N-demethylation activity was assessed in the supernatants by photometrical determination of the reaction product formaldehyde (12). For this reaction, the supernatants were diluted 1:4 with 0.1 M sodium phosphate buffer pH 7.4. Reactions were performed in a shaking water bath for 10 min at the temperatures indicated (26°, 32°, 38°, 44°C). Before starting the reaction with the substrate ethylmorphine, samples were allowed to equilibrate to the temperature for 5 min. The activities of the model reactions were referenced to the protein content of the supernatants.
Values were presented as means ± sd. One-way analysis of variance with repeated measures was performed within the group. Post hoc comparisons were done using paired Student's t-test with Bonferroni correction for multiple comparisons. Differences were considered significant at P < 0.05. All statistical tests were done using the statistical package SPSS for Windows release 10.0 (SPSS Inc., Chicago, IL).
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Results |
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o2 was reduced by 44% ± 11% (P < 0.05) and returned to prehypothermic values after rewarming.
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The baseline plasma fentanyl concentration did not change during the prehypothermic period (MP1, MP2) (Fig. 2). At the end of the cooling period, the plasma fentanyl concentration increased by 25% ± 11% (MP3, P < 0.001). After rewarming (MP4) and 6 h of normothermia (MP5), plasma fentanyl concentration remained increased (P < 0.05).
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As shown in Figure 3, hypothermia was associated with markedly reduced blood flows (P < 0.05) to the kidney (38% ± 32%), spleen (45% ± 33%), stomach (53% ± 31%), and gut (49% ± 26%). Pancreatic blood flow was also reduced during hypothermia (49% ± 46%), and remained so even after rewarming (P < 0.05). In contrast, hepatic artery blood flow remained unchanged throughout the experiment.
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Hypothermia induced a strong temperature-dependent reduction in hepatic CYP3A4 activity (at 26°C: 48% ± 2%, at 32°C: 69% ± 1%, compared with values obtained at 38°C) (P < 0.001). Temperature increase was associated with a mildly reduced CYP3A4 activity (at 44°C: 94% ± 3%) (P < 0.01) (Fig. 4).
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Discussion |
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We chose a porcine model because of the similarities between swine and humans in the basic cardiovascular variables, including CI and o2, as well as regional distribution of blood flows (17). Furthermore, there is a remarkable similarity in biotransformation pathways, because the activity of the most important CYP isoform in humans, CYP3A4, also responsible for hepatic fentanyl metabolism, is present in pigs with comparable levels and activities (9).
Under normal conditions, fentanyl is cleared predominantly by hepatic biotransformation (7), and the metabolism is extensive and rapid under physiological conditions. Furthermore, fentanyl is preferentially oxidized to norfentanyl by hepatic microsomal cytochrome P450 3A isoform (8) and excreted renally. Other pathways such as intestinal metabolism seem less likely with parenteral administration (18).
There are several processes that may be responsible for an increase in plasma fentanyl concentration during hypothermia. The pharmacokinetics of fentanyl, a drug with a large distribution volume and a high hepatic extraction ratio, could be altered. After a bolus injection, distribution volume and total body clearance were markedly reduced during hypothermia (14). A reduced total body clearance can be a result of reduced hepatic biotransformation and/or slower distribution caused by reduced cardiac output and organ perfusion rate estimated in this study. Simulation studies have demonstrated that the amount of reduced perfusion reported in this study can be responsible for increased blood fentanyl concentration (19). However, the experimental design does not allow discrimination between alterations in distribution and elimination influences.
With regard to hypothermia-dependent alterations of fentanyl elimination, several aspects have to be considered. We have shown that hepatic CYP3A4 activity in juvenile pigs is strongly temperature dependent. At 32°C, the conversion rate was reduced by about one-third. Therefore, a relevant component of the reduction in total body clearance seems to be a reduced hepatic biotransformation of fentanyl. Furthermore, total hepatic blood flow is assumed to be reduced, despite the surprising finding that only arterial hepatic influx remained unaltered during mild hypothermia. Under normal conditions, arterial hepatic blood flow represents no more than one-fifth of total hepatic blood flow in the juvenile pig (20). The other influx arrives via the portal vein. Indeed, there is no doubt that portal blood flow was markedly reduced during hypothermia, because all perfusion rates of splanchnic organs drained by the portal vein were reduced. Because fentanyl has a high liver extraction ratio, hepatic elimination of fentanyl is expected to be more sensitive to blood flow alterations than to enzymatic activity (21). We suggest that both mechanisms, i.e., reduced total liver blood flow and reduced hepatic CYP3A4 activity, are involved in the reduced hepatic elimination, which may in turn be responsible for the increased efficient levels of fentanyl during hypothermia.
Hemodynamic data support our opinion that hypothermia-dependent alteration of fentanyl turnover is not primarily caused by compromised hepatic energy metabolism. The expected effects of mild hypothermia on systemic hemodynamics and oxygen uptake were similar to that reported earlier with a comparable anesthetic/analgesic regimen (22). Our data demonstrate a marked decrease in CI (CI decreased by an average 41%), and concomitant decrease in whole body O2 uptake which suggests an appropriate decreasing of metabolic demand. This assumption is supported by an unaltered arteriovenous oxygen extraction rate during and after mild hypothermia (Table 1). Furthermore, during hypothermia, similar reductions in blood flow have been shown for the splanchnic organs studied, suggesting portal blood flow to the liver appropriate to a reduced oxidative metabolism. The maintained hepatic artery influx tends to exclude hepatic hypoperfusion and restricted hepatic O2 availability for the reduced fentanyl metabolism.
Thus, a risk for increased blood concentrations during long-term administration of fentanyl depends mainly on portal blood flow, which is proportional to cardiac output. Hence, maintenance of cardiac output to normal values may suggest that fentanyl concentration is likely stable whereas a decrease in cardiac output should suggest decreasing fentanyl doses.
The reason for a delayed normalization of plasma fentanyl level after rewarming remains unclear. A prolonged inhibition of hepatic CYP3A4 activity seems rather implausible, because after long-term deep hypothermic liver preservation, hepatic drug extraction was reestablished within 30 min of rewarming (23). An augmented tissue accumulation of fentanyl during the hypothermic period of reduced clearance, and subsequent rebound of plasma levels after rewarming increases tissue blood flow, may be involved.
Additionally, it should be noted that hepatic CYP3A4 is also responsible for midazolam biotransformation, hence this drug could also accumulate during mild hypothermia. In this study, however, we did not estimate plasma midazolam levels. There is no information about the effects of hypothermia on midazolam pharmacology. Nevertheless, it is quite possible that a decreased midazolam metabolism during hypothermia might have resulted in increased midazolam concentration with a possible competition on fentanyl metabolism. Provided that a similar response occurs as with fentanyl, a prolonged accumulation of two drugs could overcome the transforming capacity of CYP3A4 for xenobiotics, resulting in a delayed normalization of the respected plasma levels. Furthermore, midazolam could presumably enhance the hemodynamic effect of hypothermia on regional blood flow and fentanyl transport to the liver. A biphasic response of portal blood flow to bolus administration of midazolam has been reported with an early increase and a following decrease. This is probably related to redistribution of blood within the splanchnic system (blood mobilization from spleen and intestine), whereas hepatic arterial flow decreased immediately after administration (24).
Although the underlying mechanisms of the persistent increase of plasma fentanyl concentration remain speculative, the description of the phenomenon may be of clinical relevance with respect to the recovery from sedation of intensive care unit (ICU) patients after therapeutic hypothermia. However, the range of concentrations usually required in ICU can be smaller than in anesthesia. Therefore, the consequences of hypothermia-related fentanyl accumulation per se seem less relevant. However, hypothermia can aggravate a fentanyl overdosage during continuous long-term administration and can result in negative side effects such as decreased intestinal motility, hypotension, reduced tissue extraction capabilities, prolonged ICU stay, increased costs, and acute withdrawal syndrome (25). Consequently, an optimal analgesic/sedative regimen during the application of hypothermia should be carefully chosen.
Nevertheless, we can conclude that hypothermia of 32°C induces a reduction of CI, hypoperfusion in several organs, and an increase in plasma fentanyl concentrations. With rewarming, there is a prolonged recovery phase which may include increased side effects and prolonged recovery from analgesia/sedation.
The authors acknowledge the advice of U. Jaeger, I. Witte, and L. Wunder and their skillful technical assistance (Institute for Pathophysiology and Pathobiochemistry, University Jena, Germany), and Don Bredle, PhD (Department of Kinesiology, University of Wisconsin, Eau Claire, WI), for advice in preparing the manuscript.
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Footnotes |
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Accepted for publication September 8, 2004.
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