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Anesth Analg 2003;96:1350-1353
© 2003 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Halothane and Isoflurane Have Additive Minimum Alveolar Concentration (MAC) Effects in Rats

Edmond I Eger, II, MD*, Yilei Xing, MD*, Michael Laster, DVM*, James Sonner, MD*, Joseph F. Antognini, MD{dagger}, and Earl Carstens, PhD{dagger}

*Department of Anesthesia and Perioperative Care, University of California, San Francisco, and {dagger}Department of Anesthesiology and the Section of Neurobiology, Physiology, and Behavior, University of California, Davis

Address correspondence and reprint requests to Edmond I Eger II, MD, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to egere{at}anesthesia ucsf.edu.


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Studies suggest that at concentrations surrounding MAC (the minimum alveolar concentration suppressing movement in 50% of subjects in response to noxious stimulation), halothane depresses dorsal horn neurons more than does isoflurane. Similarly, these anesthetics may differ in their effects on various receptors and ion channels that might be anesthetic targets. Both findings suggest that these anesthetics may have effects on movement in response to noxious stimulation that would differ from additivity, possibly producing synergism or even antagonism. We tested this possibility in 20 rats. MAC values for halothane and (separately) for isoflurane were determined in duplicate before and after testing the combination (also in duplicate; six determinations of MAC for each rat). The sum of the isoflurane and halothane MAC fractions for individual rats that produced immobility equaled 1.037 ± 0.082 and did not differ significantly from a value of 1.00. That is, the combination of halothane and isoflurane produced immobility in response to tail clamp at concentrations consistent with simple additivity of the effects of the anesthetics. These results suggest that the immobility produced by inhaled anesthetics need not result from their capacity to suppress transmission through dorsal horn neurons.

IMPLICATIONS: Despite differences in their capacities to inhibit spinal dorsal horn cells, isoflurane and halothane are additive in their ability to suppress movement in response to a noxious stimulus.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
MAC is the minimum alveolar concentration required to eliminate movement in response to noxious stimulation in 50% of subjects (an anesthetic ED50). We have shown that 0.9–1.1 MAC halothane, but not 0.7–1.4 MAC isoflurane, depresses the response of dorsal horn neurons to noxious thermal stimuli (1). These results confirm earlier results indicating that 0.7–1.1 MAC isoflurane minimally affects the response of dorsal horn neurons to stimulation (2,3). The differential effect of isoflurane and halothane on neuronal responses to noxious thermal stimulation suggests different sites of action for these two anesthetics. The results suggest that halothane acts, at least partially, at dorsal horn neurons, whereas isoflurane acts at more ventral sites (e.g., motoneurons). Movement that follows noxious stimulation results from transmission of impulses from the periphery to dorsal horn neurons and, via one or more synapses, to motoneurons. Drug effects at two different sites (or two different receptors) could result in synergism, i.e., where the effect produced by a combination of two drugs results in a greater effect than either drug alone. This has been describ-ed for gamma-aminobutyric acid (GABA)-agonism/glutamate-antagonism (4), {alpha}-adrenergic/opiate (5), and midazolam/isoflurane interactions (6). Thiopental and halothane seem to act synergistically (7). Although halothane and isoflurane are generally thought to have similar actions at various receptors and ion channels that are potential sites of anesthetic action (8), differing effects of halothane and isoflurane at various receptors (e.g., GABA and glutamate) and ion channels (e.g., K channels) have been described (9–11). These differences might underlie the divergent effects we have reported (12) insofar as receptors are differentially distributed in the spinal cord and brain (13). Such differences in the effects of halothane and isoflurane suggest that, unlike many other combinations of potent inhaled anesthetics (14), isoflurane and halothane might be synergistic in their capacities to suppress movement in response to noxious stimulation. The present report tested this possibility.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
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With approval of the Committee on Animal Research of the University of California, San Francisco, we studied 20 male specific-pathogen-free Sprague-Dawley rats (Crl:CD(SD)BR) weighing 300–450 g obtained from Charles River Laboratories (Hollister, CA).

MAC for halothane, isoflurane, and the combination of halothane and isoflurane was determined in duplicate (i.e., six separate experiments were performed on each rat on separate days, and each experiment was separated from the next by a minimum of 3 days). MAC was determined in 10 rats concurrently. Each rat was placed in an individual gas tight clear plastic cylinder closed at both ends by rubber stoppers. The stoppers were pierced with holes for various purposes. A rectal temperature probe (temperature maintained between 36°C and 38.5°C) and the rat’s tail were separately drawn through holes in the rubber stopper closing the distal end of the cylinder. Delivered gases at an average inflow rate of 1 L/min to each rat entered through ports at the head (proximal) end of the cylinder and exited at the tail (distal end), a flow sufficient to minimize rebreathing (inspired CO2 <10 mm Hg). Exiting gases were scavenged.

Isoflurane and halothane were introduced from conventional vaporizers. For the determination of MAC for the individual anesthetics, the initial concentration was set at a concentration that permitted movement of all rats in response to noxious stimulation. Animals were exposed to the initial concentrations for 30 min for isoflurane and 40 min for halothane or when a combination of isoflurane and halothane was administered. A tail clamp was then applied for 1 min or until the animal moved, and the anesthetic partial pressure was measured by gas chromatography. If the animal moved, the partial pressure was increased by approximately 0.2% atmospheres of halothane or approximately 0.3% atmospheres of isoflurane. After equilibration for 30 min, the tail clamp was applied again and the anesthetic partial pressure measured by chromatography. This procedure was repeated until the partial pressures bracketing movement-nonmovement were determined for each rat.

For the determination of the MAC of the combination, the initial concentration of one anesthetic was set at half the average MAC value for the population, and the initial concentration of the other anesthetic was set at approximately 20% of the average MAC value for the population. This was reversed for the second determination of the MAC of the combination. Thus, we began with what would be 0.7 MAC (a sum of 0.5 plus 0.2 MAC) if the anesthetics were additive in their effects, and all rats moved in response to tail clamp at this combination of concentrations. The concentration of the anesthetic that initially was 0.2 MAC was then increased by approximately 0.2 MAC steps of that anesthetic until all rats failed to move in response to clamp of the tail.

Anesthetic concentrations for individual anesthetics and CO2 were monitored with an infrared analyzer (Datascope, Helsinki/Finland), but the concentration used in determinations of MAC was obtained using gas chromatography. We used a Gow-Mac gas chromatograph (Gow-Mac Instrument Corp, Bridgewater, NJ) equipped with a flame ionization detector to measure isoflurane and halothane concentrations. The 4.6-m-long, 0.22-cm (ID) column was packed with SF-96. The column temperature was 138°C–151°C. The detector was maintained at temperatures approximately 50°C warmer than the column. The carrier gas flow was nitrogen at a flow of 15–20 mL/min. The detector received 35–38 mL/min of hydrogen and 240–320 mL/min of air. We commonly used secondary (cylinder) standards referenced to primary (volumetric) standards.

MAC was defined as the average of the partial pressures that just prevented and permitted movement in response to clamping the tail. Two MAC values were obtained for a given rat for each anesthetic both before and after testing the combination, and the four determinations were averaged to provide the individual rat values for MAC for isoflurane and halothane. For the combination of halothane and isoflurane, the concentrations of isoflurane and halothane just permitting and just preventing movement in response to the tail clamp were divided by the MAC values for that animal, thereby giving the MAC fraction contributed by each anesthetic. These MAC fractions were used to construct an isobologram with the MAC fraction for halothane on one axis and that for isoflurane on the other axis. In addition, the MAC fractions for a given rat were averaged and the averages compared with the value of 1.0 (paired Student’s t-test) to assess whether the results deviated from additivity. We accepted P < 0.05 as indicating significance.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
One rat died between the fifth and sixth determinations of MAC, and the value for the sixth determination for this rat was assumed to equal that of the fifth determination. The combination of isoflurane and halothane produced an additive effect rather than either antagonism or synergism (Fig. 1; Table 1). The sum of the MAC-fraction contribution of each anesthetic equaled 1.037 ± 0.082, a value not significantly different from 1.000 (P > 0.05).



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  		Figure 1. Minimum alveolar concentration (MAC) for isoflurane and (separately) for halothane was determined for each of 20 rats. The combination of halothane and isoflurane that bracketed movement-nonmovement was determined. The concentrations of isoflurane and halothane are plotted in the present isobologram as fractions of the MAC for each. The data lie on the line of additivity, showing neither synergism nor antagonism.

 

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  		Table 1. Halothane and Isoflurane, Alone and in Combination, Required to Produce Immobility in 50% of Rats
 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our results do not support the prediction that the combination of halothane and isoflurane should produce results for MAC that deviate from additivity. Thus, either the differential depression of dorsal horn neurons (MAC of halothane depresses more than MAC of isoflurane to noxious thermal or other stimuli (2,3,12)) is not relevant to the capacity of these anesthetics to suppress movement in response to noxious stimulation or other actions precisely balance the differential effects on dorsal horn neurons. A similar interpretation may be made concerning the differential effects (albeit, often only slightly different) of these anesthetics on receptors and channels affected by isoflurane and halothane (e.g., GABA-R (15), Glu-R (16), and potassium channels (11)). A parsimonious interpretation of our data is that, to produce immobility, halothane and isoflurane act in common on some site other than dorsal horn neurons or receptors and channels that might be differentially affected by isoflurane versus halothane.

Synergy can result from drug action at two different anatomic sites or two different receptor types. Opiate effects at supra-spinal sites can act synergistically with opiate actions at the spinal cord to produce antinociception; this presumably occurs as the result of descending inhibition on pain pathways in the spinal cord (17). Demonstration of synergism, however, depends on the type of nociceptive stimulus (18). We did not test whether another nociceptive test (e.g., tail flick) might have permitted detection of synergism. Antinociceptive effects obtained using one test (e.g., tail flick latency) cannot be extrapolated to an effect on MAC where a supramaximal stimulus is used. For example, Joo et al. (19) found that knockout of the glutamate-R2 subunit altered anesthetic effects on the hind paw withdrawal latency but not on MAC.

In addition to any differential effect within the spinal cord, we cannot exclude differential effects elsewhere in the central nervous system. For example, Kingery et al. (20) found that isoflurane had a supraspinal pronociceptive effect, in addition to a supra-spinal and spinal antinociceptive effect. It is unknown if halothane has a similar action. We cannot exclude the possibility that isoflurane’s pronociceptive effect fortuitously and precisely countered a local synergistic effect of halothane and isoflurane at the spinal cord. Kingery et al. (20) determined that the pronociceptive effect was present at 0.8%–1.2% isoflurane; data at other isoflurane concentrations were not described. The authors did not report MAC in their animals, but presumably this represents 0.5–0.8 MAC, a range in which some of our animals were tested.

Stone et al. (7) examined the effects of thiopental on halothane MAC. Although they did not perform a formal isobolographic analysis, their data suggest that thiopental and halothane interact synergistically. They found that thiopental had a calculated effective dose 50% (ED50) of about 46 µg/mL and that a thiopental concentration of about 7 µg/mL reduced halothane MAC 50%, consistent with a synergistic effect. It is noteworthy that thiopental plasma concentrations in this range are associated with hyperalgesia (21). This suggests that, in the present study, if isoflurane did have a pronociceptive effect, it might have been masked by direct local effects. Finally, thiopental and halothane clearly have different actions at different anatomic sites. We have shown that thiopental’s action in the brain to suppress movement is more potent than that of halothane and isoflurane (22). Such divergent anatomic sites of action, combined with thiopental’s synergistic effect on halothane MAC (7), supports the interpretation of our data that any difference between halothane and isoflurane regarding anatomic sites of anesthetic action is small, and not important for MAC.

What clinical implications might follow from the present study? Combinations of potent inhaled anesthetics, particularly azeotropic mixtures (constant boiling mixtures of the combination of two liquid anesthetics producing constant fractions of each anesthetic in the output of a vaporizer containing the azeotrope) have been suggested for clinical use (23–26). The combination of diethyl ether and halothane has been most studied. Would the combination of desflurane and sevoflurane produce an anesthetic superior to each alone, e.g., one with a very rapid awakening and no respiratory irritant effects? Our data suggest, however, that such combinations would produce additive anesthetizing effects.


   Acknowledgments
 
Supported, in part, by NIH grant 1PO1GM47818–08 and GM61283.


   Footnotes
 
Dr. Eger is a paid consultant to Baxter Healthcare Corp. Baxter contributed the isoflurane used in these studies.


   References
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 Introduction
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 Results
 Discussion
 References
 

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Accepted for publication December 19, 2002.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2003 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press